Human hyaluronan receptor

ABSTRACT

The invention provides the genomic and cDNA sequences of human RHAMM as well as diagnostic and prognostic tests for malignancy in humans.

[0001] The present invention relates to the human hyaluronan receptor,known as the Receptor for Hyaluronic Acid Mediated Motility or RHAMM.More particularly, it relates to the genomic and cDNA sequences of thehuman hyaluronan receptor.

BACKGROUND OF THE INVENTION

[0002] In the description which follows, references are made to certainliterature citations which are listed at the end of the specification.

[0003] Hyaluronan is a large glycosaminoglycan that is ubiquitous in theextracellular matrix and whose synthesis has been linked to cellmigration, growth and transformation. This glycosaminoglycan interactswith cell surfaces via specific protein receptors that mediate many ofits biological effects.

[0004] One of these receptors is RHAMM. A RHAMM cDNA was originallycloned from a murine 3T3 fibroblast cDNA expression library (Hardwick etal., (1992) and several RHAMM isoforms were found to be encoded withinthe murine gene (Entwistle et al., (1995).

[0005] RHAMM acts downstream of ras in the ras transformation pathway(Hall et al., 1995). It regulates focal adhesion turnover, is requiredfor cell locomotion and is transforming when overexpressed in murinecells (Hall et al., 1995).

SUMMARY OF THE INVENTION

[0006] In accordance with one embodiment of the invention, an isolatednucleic acid comprises a nucleotide sequence encoding a protein selectedfrom the group consisting of human RHAMM 1, human RHAMM 2, human RHAMM3, human RHAMM 4 and human RHAMM 5.

[0007] In accordance with a further embodiment of the invention, anisolated nucleic acid comprises a nucleotide sequence selected from thegroup consisting of

[0008] (a) a nucleotide sequence of at least 10 consecutive nucleotidesof Sequence ID NO:3;

[0009] (b) a nucleotide sequence of at least 15 consecutive nucleotidesof Sequence ID NO:3; and

[0010] (c) a nucleotide sequence of at least 20 consecutive nucleotidesof Sequence ID NO:3.

[0011] In accordance with a further embodiment of the invention, anisolated nucleic acid comprises a nucleotide encoding at least onebinding domain of human RHAMM protein or a fragment or analogue thereofwhich retains HA binding ability.

[0012] In accordance with a further embodiment of the invention, anisolated nucleic acid comprises a nucleotide sequence of at least oneexon of the nucleotide sequence of Table 1.

[0013] In accordance with a further embodiment of the invention, anisolated nucleic acid comprises a nucleotide sequence encoding the aminoacid sequence of Sequence ID NO:50.

[0014] In accordance with a further embodiment, the invention provides atransgenic animal wherein a genome of the animal, or of an ancestorthereof, has been modified by insertion of at least one recobinantconstruct to produce a modification selected from the group consistingof

[0015] (a) insertion of a nucleotide sequence of at least one exon ofthe human RHAMM gene;

[0016] (b) insertion of a nucleotide sequence encoding at least onehuman RHAMM protein;

[0017] (c) inactivation of an endogenous RHAMM gene.

[0018] In accordance with a further embodiment, the invention provides asubstantially pure protein selected from the group consisting of humanRHAMM 1, human RHAMM 2, human RHAMM 3, human RHAMM 4 and human RHAMM 5.

[0019] In accordance with a further embodiment, the invention provides asubstantially pure peptide comprising an amino acid sequence selectedfrom the group consisting of

[0020] (a) at least 5 consecutive amino acid residues from the aminoacid sequence of Sequence ID NO:4;

[0021] (b) at least 10 consecutive amino acid residues from the aminoacid sequence of Sequence ID NO:4; and

[0022] (c) at least 15 consecutive amino acid residues from the aminoacid sequence of Sequence ID NO:4.

[0023] In accordance with a further embodiment of the invention, asubstantially pure peptide comprises at least one binding domain ofhuman RHAMM.

[0024] In accordance with a further embodiment, the invention provides asubstantially pure peptide having the amino acid sequence of Sequence IDNO:50.

[0025] In accordance with a further embodiment, the invention providesan antibody which selectively binds to an antigenic determinant of ahuman RHAMM protein.

[0026] In accordance with a further embodiment, the invention providesan antibody which selectively binds to an antigenic determinant of thepeptide of Sequence ID NO:50.

[0027] In accordance with a further embodiment, the invention provides amethod for identifying compounds which can selectively bind to a humanRHAMM protein comprising the steps of

[0028] providing a preparation of at least one human RHAMM protein;

[0029] contacting the preparation with a candidate compound; and

[0030] detecting binding of the RHAMM protein to the candidate compound.

[0031] In accordance with a further embodiment, the invention provides amethod for assessing prognosis in a mammal having a tumour, comprisingobtaining a tumour sample from the mammal and determining the level ofexpression of RHAMM protein in the tumour sample, wherein increasedexpression of RHAMM protein is indicative of a poor prognosis.

[0032] In accordance with a further embodiment, the invention provides apharmaceutical composition for preventing or treating a disorder in ahuman characterised by overexpression of the RHAMM gene comprising aneffective amount of a nucleotide sequence selected from the groupconsisting of

[0033] (a) a dominant suppressor mutant of the RHAMM gene;

[0034] (b) an antisense sequence to human RHAMM cDNA; and

[0035] (c) an antisense sequence to exon 8 of the human RHAMM gene and apharmaceutically acceptable carrier.

[0036] In accordance with a further embodiment, the invention provides amethod for preventing or treating a disorder in a human characterised byoverexpression of the RHAMM gene comprising administering to the mammalan effective amount of a nucleotide sequence selected from the groupconsisting of

[0037] (a) a dominant suppressor mutant of the RHANN gene;

[0038] (b) an antisense sequence to human RHAMM cDNA; and

[0039] (c) an antisense sequence to exon 8 of the human RHAMM gene.

[0040] In accordance with a further embodiment, the invention provides amethod for inhibiting cell migration in a human comprising administeringto the human an effective amount of an agent selected from the groupconsisting of

[0041] (a) an antibody which binds specifically to human RHAMM proteinor a fragment thereof; and

[0042] (b) a peptide comprising a human RHAMM HA-binding domain.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Certain embodiments of the invention are described, referencebeing made to the accompanying drawings, wherein:

[0044]FIG. 1 shows the strategy used for cloning human RHAMM cDNA. Thecoding region of the human RHAMM cDNA is represented by an openrectangle, the start (ATG) and stop (TAA) codons are indicated, as arethe 5′ and 3′ UTRs. The nucleotide region encoded in each clone andRT-PCR product is indicated by a single line.

[0045]FIG. 2 shows a comparison of the amino acid sequences of theHA-binding domains of mouse, rat and human RHAMM. Specifically-spacedbasic amino acids corresponding to the termini of the various consensushyaluronan-binding motifs, X¹—A_(n)—X², contained within the bindingdomains are underlined in the mouse sequence, amino acids 402 to 412(Sequence ID NO:1) and amino acids 424 to 433 (Sequence ID NO:2), thenumbering indicating the amino acid position of the HA-binding domainswithin the published amino acid sequence of mouse RHAMM2 (Hardwick etal., 1992). In the rat and human binding domains, amino acids identicalto the mouse sequence are represented by dots.

[0046]FIG. 3 shows immunohistochemical staining of formalin-fixedparaffin-embedded human breast cancer tissues using an antibody toRHAMM. Sections are counterstained with methyl green. The stainingintensity of tumor cells and stroma is variable. A, B, & C, Generaltumor staining (arrow heads) with maximum tumor staining in individualcells (arrows), D. Tumour cells and stroma both staining positively forRHAMAM, E. Tumours showing positive nuclear as well as cytoplasmicstaining, and F. Tumours showing negative staining. Magnification, A andF, 400×; B,D and E, 250×; C, 650×.

[0047]FIG. 4 shows Kaplan-Meier survival curves of primary breast cancerpatients subdivided according to RHAMM maximum staining.

[0048]FIG. 5 shows Kaplan-Meier survival curves for overall survival ofprimary breast cancer patients. The top two curves are for node negativeand the bottom two curves are for node positive patients. Open symbolsare for tumors with maximum-general RHAMM staining<1 unit; closedsymbols are for tumors with values≧1 unit.

[0049]FIG. 6 shows Kaplan-Meier survival curves for metastasis-freesurvival of primary breast cancer patients. The top two curves are fornode negative and the bottom two curves are for node positive patients.Open symbols are for tumors with maximum-general RHAMM staining<1 unit;closed symbols are for tumors with values≧1 unit.

[0050]FIG. 7 shows in diagrammatic form the presence or absence of exons7 and 8 in human RHAMM isoforms 1 to 5.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The inventors have obtained the genomic sequence for human RHAMMshown in Table 1. The human RHAMM gene spans 25.4 Kilobases andcomprises 17 exons.

[0052] The inventors have also obtained and sequenced the full lengthcDNA for human RHAMM. The cDNA, from normal human breast, has a2175-nucleotide open reading frame (Sequence ID NO:3), which encodes apolypeptide of 725 amino acids (Sequence ID NO:4), corresponding to amolecular weight of 84 kDa.

[0053] Western analysis of the normal human breast cell line, MCF-10A,using as probe antibody R3, an antibody to murine RHAMM aa⁴²⁵⁻⁴⁴³,demonstrated three specific RHAMM protein bands of 84, 70 and 60 kDa.The major protein, which had molecular weight 70 kDa, may be generatedby alternative splicing or post translational modification of themessage encoding the 84 kDa protein. Also, the second ATG codon (+346,aa 116) has a perfect Kozak configuration and may be preferentially usedin vivo resulting in a 70 kDa protein. Alternatively, the human RHAMMcDNA may correspond to the minor 84 kDa protein species, a possibilitysuggested by the observation that murine RHAMMv4 is expressed at lowamounts in nontransformed cells (Entwistle et al., 1995). The aboveresults and the presence of a stop codon in the 5′ noncoding region,in-frame with the initiation methionine, in both the human RHAMM cDNAand RT-PCR product, indicate that the cDNA is full length.

[0054] The human RHAMM protein has been found to occur in severalisoforms, shown diagrammatically in FIG. 7. Similar isoforms have beenidentified in the mouse. The longest isoform, corresponding to thecomplete cDNA, is designated RHAMM 5. A shorter version of this protein,lacking the signal peptide seen in RHAMM5, is designated RHAMM 4.

[0055] Both RHAMM 4 and RHAMM 5 include exons 7 and 8. Alternativelyspliced isoforms 1 and 3 lack exon 8 and exon 7 respectively. Theshortest isoform, RHAMM 2, lacks both exon 7 and exon 8 and correspondsto the first described mouse RHAMM 2.

[0056] Table 2 shows a comparison of the full length human RHAMM cDNAand murine RHAMM 4 cDNA (Sequence ID NO:5), identical nucleotides beingindicated by vertical broken lines, nucleotide gaps required to maintainalignment being indicated by a dash and start and stop codons beingshown in bold.

[0057] Table 3 shows a comparison of the human RHAMM amino acid sequenceand the murine amino acid sequence (Sequence ID NO: 6) encoded by thenucleotides of Table 2.

[0058] Identical amino acids are indicated by vertical broken lines andconservative changes are indicated by a plus sign. The two HA bindingdomains are shown in bold and exon 8 of the murine RHAMM 4 isunderlined. Amino acid deletions, to maintain alignment, are indicatedby a dash and the stop codon is indicated by an asterisk. The homologybetween comparable mouse and human RHAMM isoforms is 85%.

[0059] Only one of the five amino acid repeat sequences encoded inmurine RHAMM cDNA (double underlined in Tables 2 and 3) are present inhuman RHAMM cDNA.

[0060] Alternatively spliced exon 8 has been shown to be critical to thefunction of RHAMM in cell motility, proliferation and transformation ofmurine cells (Entwistle, 1994; Hall, 1995). A review of RHAMM expressionin human tissues has shown that most normal tissues contain human RHAMM1 isoform, and do not contain detectable RHAMM 4. In contrast, tumortissues and normal tissues responding to injury show expression of theRHAMM 4 isoform.

[0061] Alternatively spliced human exon 8 (Sequence ID NO:16) encodesthe amino acid sequence VSIEKEKIDEKSETEKLLEYIEEIS (Sequence ID NO:50).

[0062] As previously described (International Patent ApplicationWO93/21312), murine RHAMM demonstrates a consensus binding motif,X¹—A_(n)—X², wherein X¹ and X² are basic amino acid residues and A_(n)is an amino acid sequence comprising seven or eight neutral or basicamino acid residues. Several versions of this motif occur within the twomurine RHAMM binding domains, at amino acids 402 to 412 and 424 to 433of the murine RHAMM 2 amino acid sequence. As seen in FIG. 2 and Table3, this binding motif is completely conserved in the rat and human RHAMMbinding domains. For human RHAMM, the binding domains comprise the aminoacid sequence KQKIKHVVKLK (Sequence ID NO:1) and KLRCQLAKKK (Sequence IDNO: 7).

[0063] Nucleic Acids

[0064] In accordance with one series of embodiments, the presentinvention provides isolated nucleic acids corresponding to or relatingto the human RHAMM nucleic acid sequences disclosed herein.

[0065] In accordance with another series of embodiments, the presentinvention provides for isolating nucleic acids which include subsets ofthe human RHAMM sequences or their complements. Such sequences haveutility as probes and PCR primers.

[0066] Expression of RHAMM Proteins

[0067] In accordance with a further embodiment, the present inventionprovides nucleic acids in which the coding sequence for a human RHAMMprotein is operably joined to endogenous or exogenous 5′ and/or 3′regulatory regions. For example, the complete ORF for human RHAMMprotein operably joined to exogenous regulatory regions may be used forexpression of the full length human RHAMM protein. The regulatory regionmay be selected from sequences that control the expression of genes ofprokaryotic or eukaryotic cells, their viruses and combinations thereof.Such regulatory regions include for example, but are not limited to, thelac system, the trp system, the tac system and the trc system.Regulatory elements may be selected which are inducible or respressible,to allow for controlled expression of the human RHAMM gene in cellstransformed with the encoding nucleic acid. Alternatively, the codingregion may be operably joined with regulatory elements which provide fortissue specific expression of the human RHAMM gene in a selected tissue.

[0068] Only selected RHAMM isoform, or a selected portion thereof, maybe expressed by selecting the appropriate encoding nucleotide sequence.

[0069] For protein expression, eukaryotic and prokayotic expressionsystems may be generated in which the selected nucleotide sequence isintroduced into a plasmid or other vector which is then introduced intoliving cells. Prokaryotic and eukaryotic expression systems allowvarious important functional domains of the protein to be recovered asfusion proteins and then used for binding, structural and functionalstudies and also for the generation of appropriate antibodies.

[0070] Typical expression vectors contain promoters that direct thesynthesis of large amounts of mRNA corresponding to the gene. They mayalso include sequences allowing for their autonomous replication withinthe host organism, sequences that encode genetic traits that allow cellscontaining the vectors to be selected, and sequences that increase theefficiency with which the mRNA is translated. Some vectors containselectable markers such as neomycin resistance that permit isolation ofcells by growing them under selective conditions. Stable long-termvectors may be maintained as freely replicating entities by usingregulatory elements of viruses. Cell lines may also be produced whichhave integrated the vector into the genomic DNA and in this manner thegene product is produced on a continuous basis.

[0071] Eukaryotic expression systems permit appropriatepost-translational modifications to expressed proteins. This allows forstudies of the gene and gene product including determination of properexpression and post-translational modifications for biological activity,identifying regulatory elements located in the 5′ region of the gene andtheir role in tissue regulation of protein expression. It also permitsthe production of large amounts of protein for isolation andpurification, the use of cells expressing the protein as a functionalassay system for antibodies generated against the protein, the testingof the effectiveness of pharmacological agents or as a component of asignal transduction system, the study of the function of the normalcomplete protein, specific portions of the protein, or of naturallyoccurring polymorphisms and artificially produced mutated proteins. TheDNA sequence can be altered using procedures such as restriction enzymedigestion, DNA polymerase fill-in, exonuclease deletion, terminaldeoxynucleotide transferase extension, ligation of synthetic or clonedDNA sequences and site-directed sequence alteration using specificoligonucleotides together with PCR.

[0072] Once the appropriate expression vector containing a selectednucleotide sequence is constructed, it is introduced into an appropriateE.coli strain by transformation techniques including calcium phosphatetransfection, DEAE-dextran transfection, electroporation,microinjection, protoplast fusion and liposome-mediated transfection.

[0073] Suitable host cells include, but are not limited to, E. coli,pseudomonas, bacillus subtillus, or other bacilli, other bacteria,yeast, fungi, insect (using baculoviral vectors for expression), mouseor other animal or human tissue cells, or cell lines such as Cos or CHO.

[0074] Suitable methods for recombinant expression of proteins aredescribed in Sambrook et al. (1989).

[0075] Substantially Pure Proteins:

[0076] In accordance with a further embodiment, the invention providesfor substantially pure preparations of human RHAMM proteins, fragmentsof the human RHAMM proteins and fusion proteins including human RHAMMprotein fragments. The proteins, fragments and fusions have utility, asdescribed herein, for the production of antibodies to human RHAMMprotein and in diagnostic and therapeutic methods, as described herein.

[0077] The present invention provides substantially pure proteins orpeptides comprising amino acid sequences which are subsequences of thecomplete amino acid sequence of human RHAMM protein. The inventionprovides substantially pure proteins or peptides comprising sequencescorresponding to at least 4 to 5 consecutive amino acids of the humanRHAMM amino acid sequence, preferably 6 to 10 consecutive amino acids,and more preferably at least 50 to 100 consecutive amino acids, asdisclosed herein. The proteins or peptides of the invention may beisolated or purified by standard protein purification proceduresincluding gel filtration chromatography, ion exchange chromatography,high performance liquid chromatography or a RHAMM immunoaffinitypurification. For example, a protein may be expressed as a fusionprotein with glutathiones-transferase (GST) and purified by affinitypurification using a glutathione column. Human RHAMM may be expressedand purified, for example, as described for murine RHAMM in EuropeanPatent Application EPO 721012A2.

[0078] Antibodies

[0079] In accordance with a further embodiment, the invention providesantibodies which selectively bind human RHAMM protein or a portion orantigenic determinant thereof. Such antibodies may be prepared byconventional methods known to those skilled in the art.

[0080] A human RHAMM protein or a portion thereof for use in antibodyproduction may be prepared by expression of a nucleotide sequencedisclosed herein or a portion thereof, as described elsewhere herein.

[0081] For a short peptide, it may be necessary to prepare a fusionprotein comprising the selected peptide and a carrier protein, to act asantigen.

[0082] The selected RHAMM protein or peptide or fusion protein isinjected into rabbits or other appropriate laboratory animals to raisepolyclonal antibodies.

[0083] Following booster injections at weekly intervals, the rabbits orother laboratory animals are bled and their serum isolated. The serumcan be used directly or the polyclonal antibodies purified prior to useby various methods including affinity chromatography.

[0084] As will be understood by those skilled in the art, monoclonalantibodies may also be produced. A selected RHAMM protein or a peptide,coupled to a carrier protein if desired, is injected in Freund'sadjuvant into mice. After being injected three times over a three weekperiod, the mice spleens are removed and resuspended in phosphatebuffered saline (PBS). The spleen cells serve as a source oflymphocytes, some of which are producing antibody of the appropriatespecificity. These are then fused with a permanently growing myelomapartner cell, and the products of the fusion are plated into a number oftissue culture wells in the presence of a selective agent such as HAT.The wells are then screened by ELISA to identify those containing cellsmaking binding antibody. These are then plated and after a period ofgrowth, these wells are again screened to identify antibody-producingcells. Several cloning procedures are carried out until over 90% of thewells contain single clones which are positive for antibody production.From this procedure a stable line of clones which produce the antibodyare established. The monoclonal antibody can then be purified byaffinity chromatography using Protein A Sepharose, ion-exchangechromatography, as well as variations and combinations of thesetechniques. Truncated versions of monoclonal antibodies may also beproduced by recombinant techniques in which plasmids are generated whichexpress the desired monoclonal antibody fragment in a suitable host.

[0085] Antibodies to RHAMM or to one or more of its HA binding domainsblock HA binding and inhibit cell locomotion. Since RHAMM/HA interactionis involved in oncogene- and growth factor-mediated cell locomotion,antibodies to human RHAMM, or to variants or fragments thereof whichretain HA binding ability, provide means for therapeutic intervention indiseases involving cell locomotion. These diseases include tumourinvasion, birth defects, acute and chronic inflammatory disorders,Alzheimer's and other forms of dementia, including Parkinson's andHuntington's diseases, AIDS, diabetes, autoimmune dieases, cornealdysplasias and hypertrophies, burns, surgical incisions and adhesions,strokes and Multiple Sclerosis. Other situations involving celllocomotion, in which intervention using antibodies to RHAMM or itsconstituent peptides could be employed, include CNS and spinal cordregeneration, contraception and in vitro fertilisation and embryodevelopment. Antibodies to RHAMM hve been shown to inhibit human spermmotility in vitro and also to inhibit fertilisation of hamster ova byhuman sperm in an in vitro system.

[0086] Suitable methods for creation of antibodies are described, forexample, in Antibody Engineering: A Practical Guide, Borrebaek, Ed., W.H. Freeman and Company, New York (1992) or Antibody Engineering, 2^(nd)Edition, Borrebaek, Ed., Oxford University Press, Oxford (1995).

[0087] Transformed Cells

[0088] In accordance with a further embodiment, the present inventionprovides for cells or cell lines, either eukaryotic or prokaryotic,transformed or transfected with a nucleic acid of the present invention.Such cells or cell lines are useful both for preparation of human RHAMMprotein or fragments thereof as described herein. They are also usefulas model systems for diagnostic and therapeutic techniques.

[0089] Methods of preparing appropriate vectors containing the nucleicacids of the invention and for transforming cells using those vectorsare known to those in the art and are reviewed, for example, in Sambrooket al., (1989).

[0090] Diagnostic or Prognostic Indicator in Breast Cancer

[0091] In accordance with a further embodiment tissues suspected ofmalignancy may be screened by determining whether or not RHAMM 5 isoverexpressed, overexpression being indicative of malignancy.

[0092] In accordance with a further embodiment, the present inventionprovides a method of assessing the prognosis of subjects with breastcancer.

[0093] On histological examination of breast tumours, extremely highlevels of RHAMM were noted to occur in individual cells or small foci ofcells (maximum staining). The presence of these cells was variable butcorrelated with increasing general staining for RHAMM. Moresignificantly, the presence of these unusual cells was prognostic ofpoor outcome (p−0.02). When maximum staining and general staining werecombined as a new statistical parameter (max-general), elevated RHAMMexpression significantly added to the prognostic value of nodal statusand tumor size (p=0.016 and p=0.008). The involvement of RHAMM in breastcarcinoma was further assessed by analyzing RHAMM mRNA level in a secondpatient cohort from a different geographic area. In this second study,RHAMM mRNA expression in human tissue was significantly associated withhigher tumour grade as well as with combined poor parameters (hightumour grade, ER negative and lymph node positive) (p=0.0357 andp=0.0213).

[0094] Tumour size and lymph node status have been shown to be theparameters that are significant for predicting overall survival inbreast cancer patients according to analyses based on a Cox proportionalhazard model. There appeared to be a relationship between RHAMMoverexpression generally within tumours and the appearance of single orsmall groups of cells that highly overexpress RHAMM. This relationshipcontributes to tumour progression since a combined score representingboth types of staining enhanced the prognostic value of node status andmetastasis free survival. It is likely that single cells expressing veryhigh levels of RHAMM arose from a background of cells expressing highlevels of this HA receptor.

[0095] An immunohistochemical study showed that combined general andmaximum RHAMM protein expression was related to survival predicated bylymph nodal status, but was independent of ER/PR status and tumourgrade. A second study which focused on mRNA expression yielded similarprognostic results and also a significant association with ER/PR statusand with higher tumour grade was obtained. This difference might be dueto the greater sensitivity of the RT-PCR technique to detect RHAMM usedin the second study.

[0096] Exon 8 Peptide

[0097] The peptide (Sequence ID No:50) encoded by human exon 8 (SequenceID NO:16) can be synthesised, and antibodies raised to it, byconventional methods, preferably after conjugating the peptide toanother antigen such as keyhole limpet haemocyanin. If mice areinoculated with conjugated antigen, spleen cells can be obtained andhybridomas produced, as will be understood by those skilled in the art.Screening by conventional methods can be carried out to obtain ahybridoma producing monoclonal antibodies with maximum affinity for theexon 8 peptide. The selected antibody can be used to construct aconventional ELISA, permitting screening of human serum or human tissuesfor soluble RHAMM containing the peptide coded by exon 8. Comparisonwith standard values obtained from normal patients can be used forcomparison to indicate overexpression and the presence of tumour.

[0098] Alternatively, antibodies to exon 8 could be created from phagedisplay libraries.

[0099] Alternatively, biopsy samples of human tumours can be examinedfor the level of expression of exon 8 peptide by histochemical means(paraffin sections or frozen sections), to provide an indicator oflikely prognosis. Histochemistry can be carried out by conventionalmethods, as previously described, for example, in Wang et al., 1992,using antibody to the exon 8 peptide as probe.

[0100] It has been shown that both soluble murine GST-RHAMM fusionprotein inhibits cell motility and also blocks cells in G2M of the cellcycle. The effect of the soluble fusion proteins on cell motility is dueto the hyaluronan binding domains and can be mimicked by peptides thatencode these hyaluronan binding domains. However, the effect of thesoluble protein on cell cycle block is not currently known but iscontained within RHAMM2 and is likely therefore to be the repeatedsequences.

[0101] By providing the cDNA sequence for human RHAMM isoforms, theinventors have provided a means of producing soluble human RHAMM proteinby expression of any of the human isoforms that include RHAMM 1, 2, 3, 4or 5 in conventional expression systems as described above. The solubleRHAMM isoforms may be used as a means of modulating the ratio of cellassociated RHAMM to soluble RHAMM thereby modifying the availability ofRHAMM ligands for the cell surface form of RHAMM which regulates celllocomotion and cell cycle. It is predicted that based on the murineresults RHAMM 2 would be sufficient to regulate events involving cellmotility and cell cycle. However, other RHAMM isoforms might be requiredfor regulating events in tumour progression since these additionalisoforms encode exon 7 and 8 (involved in tumorigenesis) unlike RHAMM 2which does not encode these exons. These human soluble RHAMM proteinscould be used clinically for wound repair, burns, reduction ofinflammation following transplantation, or prevention of tumour growthand metastasis. There are significant differences in the sequence of thehuman vs the murine RHAMM isoforms that require the use of the humanRHAMM cDNA's for production of soluble proteins so that an immuneresponse (which can be generated against a single amino acid change) isnot generated in humans negating the beneficial effects of the fusionprotein.

[0102] RHAMM Transgenic Animal Models

[0103] In accordance with a further embodiment, the present inventionprovides for the production of transgenic, non-human animal models forthe identification of the role of the RHAMM gene during embryogenesis,growth and development and to the understanding of the disease which thegene is responsible and/or related for the testing of possibletherapies. In the present invention, the development of a transgenicmodel for the study of the relationship between RHAMM gene expressionand malignancy and in particular breast cancer is particularlyadvantageous.

[0104] Mice are often used for transgenic animal models because they areeasy to house, relatively inexpensive, and easy to breed. Transgenicanimals are those which carry a transgene, that is, a cloned geneintroduced and stably incorporated which is passed on to sucessivegenerations. In the present invention, the human RHAMM gene may becloned and stably incorporated into the genome of an animal.Alternatively, altered portions of the gene sequence may be used such asthe RHAMM sequence which does not include exon 8, the coding regionthought responsible for the development of malignancy. In this manner,the specific function of alternatively spliced gene products may beinvestigated during animal development and initiation of malignancy inorder to develop therapeutic strategies.

[0105] There are several ways in which to create a transgenic animalmodel carrying a certain human gene sequence. Generation of a specificalterations of the human RHAMM gene sequence is one strategy.Alterations can be accomplished by a variety of enzymatic and chemicalmethods used in vitro. One of the most common methods is using aspecific oligonucleotide as a mutagen to generate precisely designeddeletions, insertions and point mutations in a DNA sequence. Secondly, awild type human gene and/or humanized murine gene could be inserted byhomologous recombination. It is also possible to insert an altered ormutant (single or multiple) human gene as genomic or minigene constructsusing wild type or mutant or artificial promoter elements. Morecommonly, knock-out of the endogenous murine genes may be accomplishedby the insertion of artificially modified fragments of the endogenousgene by homologous recombination. In this technique, mutant alleles areintroduced by homologous recombination into embryonic stem cells. Theembryonic stem cells containing a knock out mutation in one allele ofthe gene being studied are introduced into early mouse embryos. Theresultant mice are chimeras containing tissues derived from both thetransplanted ES cells and host cells. The chimeric mice are mated toassess whether the mutation is incorporated into the germ line. Thosechimeric mice each heterozygous for the knock-out mutation are mated toproduce homozygous knock-out mice.

[0106] Gene targeting producing gene knock-outs allows one to assess invivo function of a gene which has been altered and used to replace anormal copy. The modifications include insertion of mutant stop codons,the deletion of DNA sequences, or the inclusion of recombinationelements (lox p sites) recognized by enzymes such as Cre recombinase.Cre-lox system allows for the ablation of a given gene or the ablationof a certain portion of the gene sequence.

[0107] To inactivate a gene chemical or x-ray mutagenesis of mousegametes, followed by fertilization, can be applied. Heterozygousoffspring can then be identified by Southern blotting to demonstrateloss of one allele by dosage, or failure to inherit one parental alleleusing RFLP markers.

[0108] To create a transgenic mouse an altered version of the human geneof interest can be inserted into a mouse germ line using standardtechniques of oocyte microinjection or transfection or microinjectioninto stem cells. Alternatively, if it is desired to inactivate orreplace the endogenous gene, homologous recombination using embryonicstem cells may be applied as described above.

[0109] For oocyte injection, one or more copies of the normal humanRHAMM gene or altered human RHAMM gene sequence can be inserted into thepronucleus of a just-fertilized mouse oocyte. This oocyte is thenreimplanted into a pseudo-pregnant foster mother. The liveborn mice canthen be screened for integrants using analysis of tail DNA for thepresence of human RHAMM gene sequences. The transgene can be either acomplete genomic sequence injected as a YAC or chromosome fragment, acDNA with either the natural promoter or a heterologous promoter, or aminigene containing all of the coding region and other elements found tobe necessary for optimum expression.

[0110] Retroviral infection of early embryos can also be done to insertthe altered gene. In this method, the altered gene is inserted into aretroviral vector which is used to directly infect mouse embryos duringthe early stages of development to generate a chimera, some of whichwill lead to germline transmission.

[0111] Homologous recombination using stem cells allows for thescreening of gene transfer cells to identify the rare homologousrecombination events. Once identified, these can be used to generatechimeras by injection of mouse blastocysts, and a proportion of theresulting mice will show germline transmission From the recombinantline. This gene targeting methodology is especially useful ifinactivation of the gene is desired. For example, inactivation of thegene can be done by designing a DNA fragment which contains sequencesfrom a exon flanking a selectable marker. Homologous recombination leadsto the insertion of the marker sequences in the middle of an exon,inactivating the gene. DNA analysis of individual clones can then beused to recognize the homologous recombination events.

[0112] It is also possible to create mutations in the mouse germline byinjecting oligonucleotides containing the mutation of interest andscreening the resulting cells by PCR.

[0113] This embodiment of the invention has the most significantcommercial value as a mouse model for breast cancer. The role of RHAMMcan be idenitified during growth and development of mice to study itsexpression and effects on tissues with respect to malignancy. Since exon8 has been identified to be responsible for malignancy, transgenic micecarrying this exon as well as transgenic mice having the RHAMM genedevoid of exon 8 or carrying additional copies of this exon can be madeand studied with respect to malignancy and used as a model to studypossible therapies including pharmaceutical intervention, gene targetingtechniques, antibody therapies etc.

[0114] Antisense (AS) Therapy

[0115] The invention provides a method for reversing a transformedphenotype resulting from the expression of the RHAMM human gene sequencewhich includes exon 8, the exon thought responsible for transformationof cells into a malignant phenotype. Antisense based strategies can beemployed to explore gene function, inhibit gene function and as a basisfor therapeutic drug design. The principle is based on the hypothesisthat sequence specific suppression of gene expression can be achieved byintracellular hybridization between mRNA and a complementary anti-sensespecies. It is possible to synthesize anti-sense strand nucleotides thatbind the sense strand of RNA or DNA with a high degree of specificity.The formation of a hybrid RNA duplex may interfere with theprocessing/transport/translation and/or stability of a target mRNA.

[0116] Hybridization is required for an antisense effect to occur.Antisense effects have been described using a variety of approachesincluding the use of AS oligonucleotides, injection of AS RNA, DNA andtransfection of AS RNA expression vectors.

[0117] Therapeutic antisense nucleotides can be made as oligonucleotidesor expressed nucleotides. oligonucleotides are short single strands ofDNA which are usually 15 to 20 nucleic acid bases long. Expressednucleotides are made by an expression vector such as an adenoviral,retroviral or plasmid vector. The vector is administered to the cells inculture, or to a patient, whose cells then make the antisensenucleotide. Expression vectors can be designed to produce antisense RNA,which can vary in length from a few dozen bases to several thousand.

[0118] AS effects can be induced by control (sense) sequences. Theextent of phenotypic changes are highly variable. Phenotypic effectsinduced by AS are based on changes in criteria such as biologicalendpoints, protein levels, protein activation measurement and targetmRNA levels.

[0119] Multidrug resistance is a useful model for the study of molecularevents associated with phenotypic changes due to antisense effects sincethe MDR phenotype can be established by expression of a single gene mdrl(MDR gene) encoding P-glycoprotein (a 170 kDa membrane glycoprotein,ATP-dependent efflux pump).

[0120] In the present invention, mammalian cells in which the RHAMM cDNAhas been transfected and which express a malignant phenotype, can beadditionally transfected with anti-sense RHAMM DNA sequences in order toinhibit the transciption of the gene and reverse or reduce the malignantphenotype. Alternatively, portions of the RHAMM gene can be targetedwith an anti-sense RHAMM sequence specific for exon 8 which isresponsible for the malignant phenotype. Expression vectors can be usedas a model for anti-sense gene therapy to target the RHAMM geneincluding exon 8 which is expressed in malignant cells. In this mannermalignant cells and tissues can be targeted while allowing healthy cellsto survive. This may prove to be an effective treatment for malignanciesinduced by RHAMM.

[0121] Protein Therapy

[0122] Treatment of malignant disease due to overexpression of the humanRHAMM gene containing exon 8 can be performed by replacing the entiretranslated protein with a spliced protein which does not include theexon 8 protein sequence, or by modulating the function of the entireprotein sequence. Once the biological pathway of the RHAMM protein hasbeen completely understood, it may also be possible to modify thepathophysiologic pathway (eg. a signal transduction pathway) in whichthe protein participates in order to correct the physiological defect.

[0123] To replace the protein with a spliced protein, or with a proteinbearing a deliberate counterbalancing mutation it is necessary to obtainlarge amounts of pure RHAMM protein from cultured cell systems which canexpress the protein. Delivery of the protein to the effected tissues canthen be accomplished using appropriate packaging or administeringsystems.

EXAMPLES

[0124] The examples are described for the purposes of illustration andare not intended to limit the scope of the invention.

[0125] Methods of molecular genetics, protein and peptide biochemistryand immunology referred to but not explicitly described in thisdisclosure and examples are reported in the scientific literature andare well known to those skilled in the art.

Example 1

[0126] Cloning and DNA Sequencing:

[0127] A 5′-stretch normal human breast cDNA library in lambda gt11 wasobtained from Clontech (Palo Alto, Calif.) and screened using as probethe murine RHAMM 2 cDNA. Two positive clones (clones 1 & 2, FIG. 1) werePCR amplified using the 5′ and 3′ insert screening amplifiers from theλgt11 vector. The resulting 1.4 kb and 1.7 kb inserts were cloned intothe PCR™ TA vector (Invitrogen, San Diego, Calif.) and sequenced by thedideoxy chain termination method using the T7 Sequencing™ kit (PharmaciaBiotech, Uppsala, Sweden). The resulting cDNA sequence was missing theamino terminal region. Using Marathon™ cDNA amplification kit(Clontech), generated from the coding region of the human cDNA clone 1,a 1.4 kb 5′ RACE fragment was obtained from mRNA from a normal humanbreast epithelial cell line, MCF-10A (ATCC, Rockville, Md.). Thisproduct was cloned into pCR™ TA cloning vector and sequenced asdescribed above. The sequence obtained from these two sources was a 2.8kb fragment and contained an ORF of 2175 nt. The strategy used forcloning this cDNA is shown in FIG. 1.

[0128] Cell Line and Culture Condition:

[0129] The normal human breast epithelial cell line, designated MCF-10A,was obtained at passage 40 from ATCC (Rockville, Md.). The cells weregrown in Dulbecco's minimal essential medium (DMEM)/F-12 (1:1) medium)supplemented with 5% equine serum, 0.1 μg/ml cholera toxin, 10 μg/mlinsulin (Gibco BRL, Burlington, ON), 0.5 μg/ml hydrocortisone (SigmaChemical Co., St. Louis, Mo.) and 0.02 μg/ml epidermal growth factor(Collaborative Research, Inc., Palo Alto, Calif.) at 37° C. and 5% CO₂in air.

[0130] Isolation of RNA from Cells:

[0131] mRNA was extracted from 90% confluent cultures of the normalbreast epithelial cell line, MCF-10A, using the Micro-FastTrack™ kitfollowing the manufacturer's instructions. Briefly, the cells wereinitially lysed in detergent-based buffer containing RNase/ProteinDegrader, incubated at 45° C. and applied directly to Oligo (dT)cellulose for adsorption. DNA, degraded proteins, and cell debris werewashed from the resin with a high salt buffer (Binding buffer).Non-polyadenylated RNAs were washed off with a low salt buffer and thePolyA⁺RNA was then eluted in the absence of salt. Purity and quantity ofthe RNA was assessed by reading optical densities at 260 and 280 nm.

[0132] Reverse Transcription-Polymerase Chain Reaction (RT-PCR):

[0133] To confirm that the ORF of the human RHAMM cDNA obtained from thelibrary was full length, RT-PCR amplification using isolated RNA from ahuman breast epithelial cell line followed by DNA sequencing wasperformed. Reverse transcription was performed exactly as described inthe first-strand cDNA synthesis kit (Clontech) according tomanufacturer's instructions. Briefly, 1 μg messenger RNA, extracted asdescribed above, was reverse transcribed using a 16-mer oligo dT primerand 100U MMLV reverse transcriptase at 42° C. for 60 min. The total 20μl reaction was diluted to 100 μl by adding 80 μl of sterile water. 10μl of the diluted cDNA template was used In each 50 μl PCR reactionusing thermostable Taq and Pwo DNA polymerases (Boehringer-MannhelmExpand™ Long Template PCR System). TaqStart antibody (Clontech), andprimers 5′ GGATATCTGCAGAATTCGGCTTACT (Sequence ID NO:51) and 5′ACAGCAACATCAATAACAACAAGA (Sequence ID NO:52) derived from the humanRHAMM cDNA noncoding regions. PCR cycling parameters were denaturationat 94° C. for 1 min, denaturation at 94° C. for 45 sec, annealing at 60°C. for 45 sec and extension at 68° C. for 2 min. 35 cycles were usedwith a final extension time of 8 min. The PCR products were cloned intopCR™ TA cloning vector and sequenced as described above.

[0134] Western Immunoblot Analysis

[0135] The MCF-10A cells were grown in growth media and changed todefined media for 24 hours before harvest. After washing with ice coldPBS, the cells were lysed with ice cold modified RIPA lysis buffer (25mM Tris HCl, pH 7.2, 0.1% SDS, 1% Triton-X 100, 1% sodium deoxycholate,0.15 M NaCl, 1 mM EDTA) containing the protease inhibitors leupeptin (1μg/ml), phenylmethyl sulfonyfluoride (PMSF, 2 mM), pepstatin A (1μg/ml), aprotinin (0.2 TIU/ml) and 3,4-dichloroisocoumarin (200 μM) (allchemicals are from Sigma). Lysates were centrifuged at 13,000 rpm for 20min at 4° C. (Heraeus Biofuge 13, Baxter Diagnostics Corporation,Mississauga, Ontario) following 20 min incubation on ice. Proteinconcentrations of the supernants were determined using the DC proteinassay (Bio-Rad Laboratories, Richmond, Calif.) Five μg of total proteinfrom each cell lysate in SDS reducing sample buffer was loaded andseparated by electrophoresis on a 10% SDS-PAGE gel together withprestained molecular weight standards (Sigma). After transferring ontonitrocellulose membranes (Bio-Rad) in a buffer containing 25 mMTris-HCl, 192 mM glycine, 20% methanol, pH 8.3, using electrophoretictransfer cells (Bio-Rad) at 100 V for 1 hour at 4° C., additionalprotein binding sites on the membranes were blocked with 5% defattedmilk in TBST (10 nM Tris base, 150 mM NaCL, pH 7.4, with 0.1% Tween 20,Sigma). The membranes were then incubated with either the primary RHAMMantibody R3, 1:100, 1 μg/ml in defatted milk TBST) or R3.6 preincubatedwith murine fusion protein overnight at 4° C. on a gyratory shaker.After washing 3 times with TBST, the membranes were incubated withhorseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000 dilutionin 1% defatted milk in TBST) for 1 hour at room temperature and washedwith TBST, then TBS. Blotting was visualized by chemiluminescence (ECL)Western blotting detection system (Amersham International Plc.,Amersham, UK) according to the manufacturer's instructions.

Example 2

[0136] Materials and Methods:

[0137] Patients and Samples

[0138] The first cohort comprised archival materials from primaryinvasive breast carcinomas of 400 patients that had been surgicallyexcised at the Massachusetts General Hospital from 1979 to 1982. Thesewere used to determine the relationship of RHAMM protein overexpressionwith previously determined pathobiological factors and with survival.These patients continued their clinical care at Massachusetts GeneralHospital. The following information was obtained from the patient'sclinical and medical records: age at diagnosis, location or primarytumour, time to metastasis, site of metastasis, therapeuticintervention, overall survival time, and cause of death. The medianfollow-up time was 10.6 years, with a minimum of one year, a maximum of16 years, and 75% of cases having follow-up of greater than 10 years.

[0139] The second cohort comprised 98 human breast tumour specimensobtained from the NCIC-Manitoba Breast Tumour Bank. In all cases,specimens obtained for the bank have been rapidly frozen at −70° C.after surgical removal. Subsequently a portion of the frozen tissue fromeach case was processed to create formalin-fixed and paraffin embeddedtissue blocks that were matched and oriented relative to the frozentissue. These paraffin blocks provided tissue for high qualityhistological sections for pathological interpretation and assessment ofthe corresponding frozen tissue. Tumours were selected from the TumourBank database to represent a range of pathological grade (Nottinghamsystem, score 4 to 9 corresponding to low to high grade) (Elston, 1991)and estrogen receptor status (as determined by ligand binding assay).Specific frozen tissue blocks were chosen in each case on the basis ofseveral further criteria as assessed in immediately adjacenthistological sections. These criteria included a cellular content ofgreater than 30% invasive tumour cells with minimal normal lobular orductal epithelial components, good histological preservation and absenceof necrosis. The majority of tumours were primary invasive ductalcarcinomas.

[0140] Antibodies

[0141] The polyclonal antibodies used in this study, R3 and anti-fusionprotein antibody, were raised in rabbits, R3 to a specific peptide(aa⁴²⁵⁻⁴⁴³) encoded in the murine RHAMM cDNA (Hardwick, 1992) which isconserved in human RHAMM cDNA (Table 2), and anti-fusion proteinantibody to glutathione transferase (GST)-RHAMM fusion protein (Yang etal, 1993) respectively. Rabbit IgG and R3 preincubated with murine RHAMMfusion protein were used as control.

[0142] Immunohistochemistry

[0143] Routine formalin-fixed, paraffin-embedded tissues were cut into 4micron sections and mounted on poly-lysine coated slides for assessingRHAMM expression. The Avidin-biotin-peroxidase complex method was usedas previously described for CD44 staining (Yang, 1992) but with thefollowing modifications. The slides were incubated with 1.5% goat serumin 0.01M Tris-buffered saline (TBS) for 1 hour to block non-specificbinding. The primary antibody, R3 was diluted with 1.5% goat serum/TBS(1:600) and incubated on slides overnight at 4° C. Endogenous peroxidaseactivity was blocked by incubating the slides with 0.6% H₂O₂ inmethoanol (Mallinckrodt) for 30 minutes at room temperature. Thedilution of antibody was chosen by determining the dilution at which nostaining was observed for reduction mammoplasties. The slides were thenincubated with biotinylated goat anti-rabbit IgG (Vectastain ABCperoxidase kit, Vector Labs, Burlingame, Calif., 1:200 in 0.01M TBS) for1 h at room temperature, following by an avidin-biotin-peroxidasecomplex (Vectastain, Vector labs, 1:200 in 0.01M TBS) to visualize boundantibody. Between each step, the slides were washed three times with0.01M TBS. The peroxidase activity was developed by incubation in 0.05%DAB (3,3′-diaminobenzidine, Sigma) and 0.1% H₂O₂ in 0.05M TBS. Theslides were counter-stained with methyl-green. Non-immune sera as wellas antibody preabsorbed with RHAMM fusion (recombinant) protein was usedas negative control.

[0144] The extent of reactivity of human breast cancer tissues to RHAMMwas assessed by two independent and blinded observers without knowledgeof clinical outcome. The staining intensity was scored using anarbitrary scale of 0 to 4+(0=negative, 4+=strongly positive).

[0145] Four measures of staining intensity were tested. It was not knowna priori which of the four scoring measures would turn out to besignificant, nor what cut-point would be useful for any of them. Thesefour measures were: 1) general overall intensity of staining; 2) scoringof foci or isolated multiple individual cells containing the mostintense staining, referred to as “maximum staining”; 3) staining withperitumour stroma; and 4) nuclear staining. The impetus for scoring“maximal scoring” came from the custom in Surgical Pathology to conferthe overall diagnostic evaluation of a malignancy from the “worst” ormost omnious area of a slide.

[0146] Extraction of RNA

[0147] Total RNA was extracted from one to three 20 μm frozen tumoursections as described by Hiller et al (1996) using a small scale RNAextraction protocol (Tri-Reagent, Molecular Research Center, Inc.,Cincinnati, Ohio) ensuring a direct correlation between the materialanalyzed and histologically assessed cellular composition. The yieldfrom tumour sections was quantitated by spectrophotometer in a 50 μlmicrocuvette. The average yield of total RNA per 20 μm section was 4μg/cm² (+/−20% variation with cellularity) and this was associated witha consistent OD^(260/280)>1.8.

[0148] Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

[0149] Analysis:

[0150] The expression of RHAMM was assessed by RT-PCR followed byagarose electrophoresis and ethidium bromide staining to visualize thePCR products. Amplification of actin was performed in parallel tocontrol for reliability of reverse transcription of amplification. RHAMMisoform bands were then assessed by subjective scoring of band presenceand intensity (0,0.5,1,2).

[0151] Reverse transcription was performed with 100 ng total RNA with 1mM dNTP, 1 unit RNase inhibitor, 2.5 mM oligo d(T) primer, 50 units ofMMLV reverse transcriptase and 1×MMLV buffer (Gibco BRL) in a totalvolume of 10 μl of 60 minutes at 37° C. Following 5 minutes incubationat 95° C., the reaction was then diluted to 40 μl and 1 μl of the cDNA(equivalent to 2.5 ng of the input RNA) was then subjected to PCR.

[0152] PCR amplifications were conducted using 1 μl of reversetranscription mixture in a volume of 50 μl, in the presence of 10 mMTris-HCl (pH 8.3), 50 mM HCl, 1.5 mM MgCl₂, 0.2 mM dATP, 0.2 mM dCTP,0.2 mM dGTP, 0.2 mM dTTP, 100 ng of each prime, and 2 U of Taq DNApolymerase. The primers used for RHAMM were the forward primer5′-GCAAACACTGGATGAGCTTGA-3′ and the reverse prier5′-TGGTCTGCTGATCTAGAAGCA-3′. PCR cycling parameters were denaturation at94° C. for 4 min, denaturation at 94° C. for 45 sec, annealing at 60° C.for 45 sec and extension at 72° C. for 2 min. 45 cycles were used with afinal extension time of 8 min. RT-PCR products were analyzed on 1%agarose gels with ethidium bromide (200 ng/ml). The 416 bp band, and insome cases additional 266 bp band were observed. These bands were cutout for sequencing. Semi-quantitative analysis of the relative amountsof RHAMM transcripts expressed was determined by comparing theexpression of the RHAMM gene with that of human actin gene, the primersfor which were the forward primer,5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ and the reverse primer,5′-CGTCTACACCTAGTCGTTCGTCCTCATACTGC-3′. This resulted in a 838 bpfragment (Clontech).

[0153] DNA Sequencing

[0154] The DNA excised from the ethidium bromide stained agarose gel waspurified using Prep-A-Gene DNA purification systems (Bio-Rad) accordingto the manufacturer's instruction and cloned into the pCR™TA vector(Invitrogen, San Diego, Calif.). it was then sequenced by the dideoxychain termination method using the T7 Sequencing™ kit (PharmaciaBiotech, Uppsala, Sweden). A 416 bp insert corresponded to part of RHAMM4 isoform while a 266 bp insert corresponded to RHAMM 4 minus exon 13.

[0155] Statistical Methods

[0156] Student's t-test was used for comparing the effects of RHAMMantibodies and peptides on cell locomotion and collagen gel invasiondata. Kaplan-Meier survival curves were plotted for each of the scoringmeasures of Immunocytochemistry (Abacus, 1994). Differences betweensurvival curves generated by using a cut-point to divide each scoringmeasure into a dichotomous rating (0 if below and 1 above the cut-point)were tested by the logrank (Mantel-Cox) test (Lee, 1995). A Coxproportional hazard model was used to determine the significance ofmultiple factors in predicting survival. Survival Tools for Statview™was used to perform the statistical analyses (Abacus) GRAPHPAD Prism™was used to test the difference of RHAMM mRNA expression.

[0157] RHAMM Expression in Human Breast Carcinoma

[0158] The overall expression of RHAMM protein was highly variable in acohort of 400 human samples of breast carcinoma, ranging from most cellsbeing negative (−) to most cells being very strongly positive (4+)(Table 4, FIG. 3). This widespread staining in the primary tumour wasdefined as general staining (arrow heads, FIGS. 3A-C and see FIGS. 3B-Efor variability). In some tumours, RHAMM was noticably overexpressed insmall foci or in multiple individual cells within the primary tumour(FIGS. 33, 3C), arrows). In these cells RHAMM was strongly expressed inboth the cytoplasm and nucleus. Staining of these cells was defined asmaximum staining.

[0159] For the general parameter, RHAMM was observed both within tumourcells (FIGS. 3A-D) and, in fewer cases, in the extracellular milieu,i.e. the stroma surrounding the tumour (FIG. 3E, Table 4), consistentwith previous reports of the occurrence of intracellular and solubleforms in murine cells. Intracellular RHAMM appeared to be bothcytoplasmic and, in some instances, nuclear (FIG. 3D). Over 80% of the400 tumours showed no reactivity for stromal staining and nuclearstaining (i.e., score 0) as noted in Table 4. It Is interesting to notethat high level of general tumour staining and of maximum staining offoci and isolated cells were highly correlated (r=0.83). Thesecorrelations were significant (p<0.001). This result indicates that theappearance of small groups of cells exhibiting high expression of RHAMM(FIG. 3C) and increased staining for RHAMM are related events (i.e.,compare (FIG. 3A with 3B).

[0160] RHAMM Overexpression in Cell Subsets is of Prognostic Value inHuman Breast Cancer

[0161] Univariate analyses of breast carcinoma tissue sections foundnodal status (p<0.001) and tumour size (p=0.03) statisticallysignificant in the first patient cohort for predicting metastasis-freeand overall survival (Table 5). Neither type of RHAMM staining in thispatient cohort correlated with “standard” prognostic factors (tumoursize, grade, estrogen receptor status, lymph node status) (Table 5).However, RHAMM overexpression in single cells or cell subsets (maximumstaining) was a prognostic factor predicting poor outcome (p=0.02) (FIG.4).

[0162] In order to assess the relationship of both maximum (Max) andgeneral (Gen) RHAMM staining in the breast carcinoma with respect tostandard prognostic factors, lymph node positive and negative patientswere analyzed with a Cox proportional hazard model (Abacus, 1994; Lee,1995), where all factors shown in Table B were included and thendeleted, one at a time until only factors with p<0.05 remained in themodel. This model included the number of positive lymph nodes, tumorsize (classified into 3 groups: ≦2,2-5 and >5cm) as well as a combinedvalue for general and maximal staining of RHAMM. Since maximum RHAMMstaining had a negative coefficient, they were combined in a new factorwhich was defined as maximum-general (Max-Gen). When data weresegregated according to lymph node status, the Max-Gen parameter allowedfurther separation of survival curves in both groups that wassignificant at p=0.008 for overall survival (FIG. 5) and significant atp=0.016 for metastasis-free survival (FIG. 6). These results weresummarized in Table 6. The odds ratios for Max-Gen staining in thistable suggest that when the Max-Gen staining difference is ≧1 unit ineither group, the chance of recurrence is 1.40 times as large as thenthe staining difference is <1 unit. Similarly the chance of death is1.59 times as large for those tumours with differences >1 compared tothose with differences <1 unit, as seen also in FIGS. 5 and 6

[0163] RT-PCR Analysis of RHAMM Messenger RNA as Prognostic Indicator inHuman Breast Carcinoma

[0164] Immunocytochemistry analysis for RHAMM protein expression inarchival paraffin blocks showed a significant relationship between RHAMMoverexpression and survival as well as a significant but complexassociation with established prognostic parameters such as lymph nodestatus. To address this relationship further, RHAMM expression wasassessed using the more sensitive technique of reversetranscription-polymerase chain reaction (RT-PCR) of mRNA extract fromtissue sections from tumours of an independent cohort of 98 patientswhere fresh frozen tissues were available. These cases were selectedspecifically to provide a range of tumour grade and ER/PR status.

[0165] mRNA was detected in human breast cancer samples thatcorresponded to the human homologue of murine RHAMM 4. For routineanalysis of RHAMM expression, RT-PCR products using primers from exon 11and exon 14 (represented as a cDNA insert of 416 bp, see methods) wereobtained (27, 31) in all tumours. A second isoform (represented as aninsert of 266 bp) containing a deletion of exon 13 occurred in 29% oftumours. These results suggest that in human tumours RHAMM occurs asmultiple isoforms. Protein translated from the (RHAMM 4 with exon 13deletion) isoform would not be recognized by the antibody used for theimmunohistochemical analysis as this is directed to an exon 13 epitopeencoded in exon 13. Elevated expression, either of the RHAMM 4, RHAMM 4(−9) isoforms or both isoforms combined, showed a significantassociation with higher tumour grade (p=0.0466, p=0.0163, p=0.0357)(Table 7). Further analysis of subsets of patients with combinedparameters of poor prognosis (high grade/ER−ve/node+ve, n=12) versuspatients with good prognosis (low grade/ER+ve/node−ve, n=15) showed asimilar significant association of RHAMM expression with poor prognosis(p=0.0063, p=0.0085, p=0.0213) (Table 8).

Example 3

[0166] The inventors screened human pWE15 cosmid library (Clontech)using human RHAMM 5 cDNA. Clones were mapped for restriction sites andthese were lined up to match restriction sites in human RHAMM 5 cDNA.Exons were sequenced and exon/intron borders noted (Table 1).

[0167] The present invention is not limited to the features of theembodiments described herein, but includes all variations andmodifications within the scope of the claims.

REFERENCES

[0168] Abacus Concerts, Survival Tools for StatView, (1994) Berkeley:Abacus Concepts Inc.

[0169] Elston, C. W., et al., (1991), Histopathology, v. 19, pp.403-410.

[0170] Entwistle, J., Yang, B., Wong, C., Li, Q., A., B., Mowat, M.,Greenberg, A. H. and Turley E. A.: Characterization of the murine geneencoding the hyaluronan receptor RHAMM, Gene (1995), v. 163, pp.233-238.

[0171] Hall, C., Yang, B., Yan, X., Zhang, S., Turley, M., Samuel, S.,Lange, L., Wang, C., Curgen, G. D., Savani, R. C., Greenberg, A. H., andTurley, E. A., (1995), Cell, v. 82, pp. 19-28.

[0172] Hardwick, C. K., Hoare, K., Owens, R., Hohn, H. P., Hook, M.,Moore, D., Cripps, V., Austen, L., Nance, M., and Turley, E. A., (1992),J. Cell. Biol., v. 117, pp. 1343-1350.

[0173] Hiller T. et al., (1996), Biotechniques, v. 21, pp. 38-44.

[0174] Lee, E. T., (1995), Statistical Methods for Survival DataAnalysis, New York: John Wiley & Sons.

[0175] Sambrook et al., (1989), Molecular Cloning: A Laboratory Manual,2 ^(nd) Edition, Cold Spring Harbor Laboratory Press Cold Spring Harbor,N.Y.

[0176] Wang, C., et al., (1992), Histochemistry, v. 98, pp. 105-112.

[0177] Yang, B. et al., (1993), J. Biol. Chem., v. 268, pp. 8617-8623.

[0178] Yang, B., Yang, B. L., Savani, R. C., and Turley, E. A., (1994),EMBO J., v. 13, pp. 286-296. TABLE 1 EXON 1CCGCCAGTGTGATGGATATCTGCAGAATTCGGCTTACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGTGTGCCAGTCACCTTCAGTTTCTGGAGCTGGCCGTCAACATGTCCTTTCCT(A)AGGCGCCCTTGAAACGATTCAATGACCCTTCTG(INTRONXgtgcgtaagggggaaagagctgggggggggagacgccctaacgccctttgcctctttcagctcccttcttgggaggcaagcaggaggcgattttagggtcgggctggggctcattcagttgattgatttttctcaaatatgctctaagcatctgttacatgccaagcactaatcaggatgctaaggataccgcagtaaacagtctccgcccgtgggcttacattcaggcggggaatactgtcaataaacagcggtaatggagaa.....tttcaatccttagtaagaaagccatatattgcctgaatatatgatgtcatctcaaaactgcgtttgctcagttgcctgtgttcctttgacccggttgatataaagggcaagatgatattgttcttcatagagaggccttctttgtaatatcaaatggatgcaattttttacatttaaaaaaagcagtttgttaatgacatttttacatttatattcactttattatgacatgttttaacttaagatca                                             EXON 2taagtaacattagataatatattaatgttttctatttcctctag)GTTGTGCACCATCTCCAGGTGCTTATGATGTTAAAACTTTAGAAGTATTGAAAGGACCAGTATCCTTTCAGAAATCACAAAGATTTAAACAACAAAAAG(INTRONX1gtaataagatcaccaaagaacaatggttatgtgatcttataagttttaaagttatgaataacaatatttaaagatgttatagcattttttaaaatgtgaagctagaactatatttaaattttatttgatggatttatgaaagggtcaagtacagaataatgctgtcatcattacattgttatataaccaggaaaattaagcaagatacttatattgata               EXON 3tgtagctt......)AATCTAAACAAAATCTTAATGTTGACAAAGATACTACCTTGCCTGCTTCAGCTAGAAAAGTTAAGTCTTCGGAATCAA(intronxiiggtgaggagcttttatatgccagctggtttatcaagtgtatcatcaaaaacatctgaaagtattgtatttgattagaatgggttaaagtgtatgaatcaaggttataactaaatctgtaaattaatgaaatgagttatcattagaactctagcaagttttacatttctgcctaggtcattatgtttaaatgtgcccttagttcacaa                                             EXON 4ttataatggtcttcaattctcaatcacttctatgttt......)AGAAGGAATCTCAAAAGAATGATAAAGATTTGAAGATATTAGAGAAAGAGATTCGTGTTCTTCTACAGGAACGTGGTGCCCAGGACAGGCGGATCCAGGATCTGGAAACTGAGTTGGAAAAG(INTRONXX1...ttatgttcttaaaatgcattaagactttaagatgtatcataggtaaatatgattattcaaatagctagtaacattagaatatctacaagcataatgtcaaaatcagagatttttccagaaactttaggggtgattattggtagcatctccttatgttggcattctatcagtgaatcatttattatcaccttgtttttgtccagattcgtgttcttctacaggaacgtggtgcccaggacaggcggatccaggatc                    EXON 5tggaaactgagttggaaag)ATGGAAGCAAGGCTAAATGCTGCACTAAGGGAAAAAACATCTCTCTCTGCAAATAATGCTACACTGGAAAAACAACTTATTGAATTGACCAGGACTAATGAACTACTAAAATCTAAG(INTRONXXgtatctgagcctcatgataacatttacaattgaataaatataaacacgttttttagggccgggcacggtggctcacgcctgtgttcccagcattttgggaggccaaggcaggcggatcacctgaggtcgggagttcgagaccagcctgaccaacatggagaaaccctgtctctactaaaaatacaaaaattagctaggcctattggcgggcgcctgtaatctcagctactcgggaggctgaggcagaagaatcacttgaacccaggaggtggaggttgcagtgagc......taaaccagcaagtcacattaaggaaaagagggataagaacagtagactggtacagtggctcatgcctgtatttccagcattttggaaggctgagggctggagaattgcttgaggccaggagtttgagaccagcctgggcaacatatcaagaccccatctctataaacaaattgaaaaattagctaggcatggtggtggtgcacaccggtaatcccagctactcaggaagatgaggcaggaggattgattgagcccaggagtttgagattatagcgagctatgatcatgccactccactctagccgtgacagcggagcgagacttgatctcttaaaaagaaaagaaaaaaaaattaaatcaatcagtaattatggtgtaggtcaaagactgttctctctaccaaagtatattaaagtcaaaaacataaccccagtgataggtagaaaaatcaatatttctctattttaaatatgtcttagcagaaaatatttc                              EXON 6tgaattttttacgtgtttgttgtatttag)TTTTCTGAAAATGGTAACCAGAAGAATTTGAGAATTCTAGCTTGGAGTTGATGAAACTTAGAAACAAAAGAGAAACAAAGATGAGG(intron2gtgagtgctgcccttggcaggtttgctgtgtctggatctggggatcagtacaactttctca                               EXON 7tttcctaaaacaggtatctttgttgtgtag)GGTATGATGGCTAAGCAAGAAGGCATGGAGATGAAGCTGCAGGTCACCCAAAGGAGTCTCGAAGAGTCTCAAGGGAAAATAGCCCAACTGGAGGGAAAACT(intron3gtaagtgagtgaatgtgaagagaattgttaagtggaagcaattcttgatttgagtctcttcacaattattgtttactagacttaaccttctcttagtacttatctcattgcctccctccagttgccctatttctctttttaaactagaatgagccctaatcattctcaaacatgttgtgctacaaagttgtatgagtgcattacttttgtacatcttctgtattattaatgatgaggaaagatttcatgatcttatgaaagtggtcattagattgaaattgagaaacactggtataggaaattgtgatttatgcacaatcctagcctttgattttgagctttaatatacatataataaaatgtgtggatagtaagtattcagtttggtgactttagcaattgtatacacctactaaccactaccaaacaagatagaacattttcatcccttcagaaagttccttca.....#ttctactaggtaggaagtggtatctcctttgtgattttaatttgttaccatgaatgttgaccttatttttatgtgcttattgaccattttatgtgcatacaacttttgcaaggtgtctattgaagtcttttgtccatttcttgcattggacagtttggtggaggtaaacagataagtaattgaagaccaggtagtctgggacaaaagctttatgggcacacaaaatgctatttagtatgttggatgggtggggaaaccaggaagaccacaaaaagaatattatttctaacacttgggatactgtaatgaaggttctgtcatcataggtttttttgcagtatatattcagaaaactttctcacttaaataaaaattttagtcttctattttgatgtaaattgtgatttgagaaattacataaaataatagttaagagttagggctctgtagtcagcctgcctgatacaggagtatctggtacataagcattatgtaagatt                                                  EXON 8attaaataacgaaactagaatgtattaacatatgcaatttttgttttag)TGTTTCAATAGAGAAAGAAAAGATTGATGAAAAATCTGAAACAGAAAAACTCTTGGAATACATCGAAGAAATTAG(intron4gtaatatgagcagtagctttaaattgaaccttatttttttaatactcagtcattttcatcatttttctgttattttccctgtgcctaaatagatgtgctttttaagataatttgt           EXON 9tttaatgcag)TTGTGCTTCAGATCAAGTGGAAAAATACAAGCTAGATATTGCCCAGTTAGAAGAAAATTTGAAAGAGAAGAATGATGAAATTTTAAGCCTTAAGCAGTCTCTTGAGGAAAATATTGTTATATTATCTAAACAAGTAGAAGATCTAAATGTGAAATGTCAGCTGCTTGAAAAAGAAAAAG(intron5gtattacagtgtttatagttactttgtttagataagtgttacatacaacatttaggaaaaatactactatgctaaaacaaccttttaaatataattagctatactaacattttaaatataattagctatatagctatacaacagcaaaaacctgtactgcattttagaatattttactcttataatgtttgttttctgtttatttcaatacagcatattacctgtcttgattgaaatatatacagtcatataattcttgactttccactaggtagctgtgtaacaatcagtagataacacagaacaagatttgtgggttttattatttagcacatagtatatattacatggagtaatgatacaaagttcacagttttgttttcttctttggaaataccatgctaaaagcagtgtaatggaatattatgggagtccaggtttctcagtcttaatgttcttatctaattccagtattcttgatgtt               EXON 10ttgagttttctag)AAGACCATGTCAACAGGAATAGAGAACACAACGAAAATCTAAATGCAGAGATGCAAAACTTAAAACAGAAGTTTATTCTTGAACAACAGGAACATGAAAAGCTTCAACAAAAAGAATTACAAATTGATTCACTTCTGCAACAAGAGAAA(intron6gtaatttaccaccatatttttttaaactgttcattttgtgtcatacatttccctatgtctctgaacacctttaaattgtgtatatcctttgatctaccaattctatctttagagtcttatcctgaggacataatcatggatatgctgaggatttagctacgtattttcactacatgttcacctagggttatgaataatgtgggaaatgacaacagatacaaaatagggaatttttaaaaaattttctggctcattcttgtgttatttaggctatataaacattacacttaccttg......taattttatgtaatatggtgtgaaaaataatgttaatatcaaagccagttgtaaaacagatatatatatataaaaatataattttagattaagaagtttctgcatgtgcgttgcatagaaaaaagcctaagatgatatttgccacaatgttaacaaggtataggaaataatctatgaaaacaaatatgctatttctatattgttttaagtttccttgaatctgtggaatttaggtttcatccttctttatctgtacttttttttgtctccta                                                       EXON 11gtacaacctcacaatgccattccaaattattttggtggttttctgtttggatatag)GAATTATCTTCGAGTCTTCATCAGAAGCTCTGTTCTTTTCAAGAGGAAATGGTTAAAGAGAAGAATCTGTTTGAGGAAGAATTAAAGCAAACACTGGATGAGCTTGATAAATTACAGCAAAAGGAGGAACAAGCTGAAAGGCTGGTCAAGCAATTGGAAGAGGAAGCAAAATCTAGAGCTGAAGAATTAAAACTCCTAGAAGAAAAGCTGAAAGG(intron7gtttgtattaataggatctcatgttttattatgacttcagatgtatttattttgagtactttttttagtattctcttatcaatcatgtgagcgtgttaggttggattatttt......ttatacctactaccttcttcacccaaatttttaaagtaaaataagcaggaaagataagttgaagctagtagaaaaatgcattaaaaaacatgctttcgaggtaagtcataaattaggatctgagctatttagcaggtaatgcagtggtgaagatatgagctatatgattcacagtttcaaaggtaaatactattttctttcttagggtagtaattgtaggtg                                   EXON 12gcattttatctttcaattatttctttttcttag)GAAGGAGGCTGAACTGGAGAAAAGTAGTGCTGCTCATACCCAGGCCACCCTGCTTTTGCAGGAAAAGTATGACAGTATGGTGCAAAGCCTTGAAGATGTTACTGCTCAATTTGAAAG(intron8gtatttttcttgggagcctgcactcttaaatatgatgtgtgcagaaaggggtgtttaccccaggaaatatgtgagcaaagcagtcacacaaaggatgattcatactagtttaaattccataatcaccaaccgtaagtgggcatttagcattatctggtaatcttattgtatttatataattccctttataatttatagaaattcccc.....tttttttttctttgaatacacagcagatgccatgtaaactcattagtacttgcctcagaacactgaattcttacctgtgttaaatgcatgaatacattaaaaactttttagttttacttagaagtatataaagtgtcccctaatcagttatgattgtcatacgcaatagttagaaaactactttgac                     EXON 13ttttttttctttttaataag)CTATAAAGCGTTAACAGCCAGTGAGATAGAAGATCTTAAGCTGGAGAACTCATCATTACAGGAAAAAGCGGCCAAGGCTGGGAAAAATGCAGAGGATGTTCAGCATCAGATTTTGGCAACTGAGAGCTCAAATCAAGAATATGTAAG(intron9gtatatagagcaaataatggccttagaaccattaagacaatttaatgttgaaagccagctagtaactgtcccttggcttgcttttggccatcttatactgcaaattaagaatttactcagttaaaaaatgacacttcttgaagagttccttgaggtttaaagaaaaaaaaaggaaaaattaatgaaagtggctata                                    EXON 14aaatgtttagtgacctcttctctctcaaaccaaag)GATGCTTCTAGATCTGCAGACCAAGTCAGCACTAAAGGAAACAGAAATTAAAGAAATCACAGTTTCTTTTCTTCAAAAAATAACTGATTTGCAGAACCAACTCAAGCAACAGGAGGAAGACTTTAGAAAACAGCTGGAAGATGAAGAAGGAAG(intron10gtaatctatgattcgaacctgagtgccttgttaactcagttacgtga                                              EXON 15ttttttaaataactatgtttttctcaatttaattcttccatgcag)AAAAGCTGAAAAAGAAAATACAACAGCAGAATTAACTGAAGAAATTAACAAGTGGCGTCTCCTCTATGAAGAACTATATAATAAAACAAAACCTTTTCAG(intron11gtttgtcagttaggagtaaacttacttgtgtttattttagggactctttgttccctattatagtgaggacagtgactcgggttttctgcaagatcattttgctctgcacttacagtgccaatttagctcactattaaaggtttatacattttattaaattatgcataattttttcccacattattgaagtataattgacaaatttaattgacataatttttcaatggacctttgtggttttaaaaaaaa......ctcatagagaatctatggagagccctgagaatatgtgaacataccttgttttcatttgtgtttttaattttctttagtgtttatggtttatatgaaactagtaagatcaaactgttttaagtcttaactttatttaaaaaatcttttt     EXON16 cag)CTACAACTAGATGCTTTTGAAGTAGAAAAACAGGCATTGTTGAATGAACATGGTGCAGCTCAGGAACAGCTAAATAAAATAAGAGATTCATATGCTAAATTATTGGGTCATCAGAATTTGAAACAAAAAATCAAGCATGTTGTGAAGTTGAAAGATGAAAATAGCCAACTCAAATCG(intron12gtttgtaaaatgacttttcattttattaaagatattggagtgggggttattctaactataatacttaaataaaatgaatatctttggtatcagaaaaaaataactgtttatagaggaaaattgagctgtgatttagtggatttattttagagtgttgaccagatgggcattcaatgttctaaagttttctagctaccgtcttaatatatattgaaaattacttgagtaaatttgatgaattcat                                      EXON 17taagctttacatatctatttccatttgcaaa......)GAAGTATCAAAACTCCGCTGTCAGCTTGCTAAAAAAAAACAAAGTGAGACAAAACTTCAAGAGGAATTGAATAAAGTTCTAGGTATCAAACACTTTGATCCTTCAAAGGCTTTTCATCATGAAAGTAAAGAAAATTTTGCCCTGAAGACCCCATTAAAAGAAGGCAATACAAACTGTTACCGAGCTCCTATGGAGTGTCAAGAATCATGGAAGTAAACATCTGAGAAACCTGTTGAAGATTATTTCATTCGTCTTGTTGTTATTGATGTTGCTGTTATTATATTTGACATGGGTATTTTATAATGTTGTATTTAATTTTAACTGCCAATCCTTAAATATGTGAAAGGAACATTTTTTACCAAAGTGTCTTTTGACATTTTATTTTTTCTTGCAAATACCTCCTCCCTAATGCTCACCTTTATCACCTCATTCTGAACCCTTTGCTGGCTTTCCAGCTTAGAATGCATCTCATCAACTTAAAAGTCAGTATCATATTATTATCCTCCTGTTCTGAAACCTTAGTTTCAAGAGTCTAAACCCCAGATTCTTCAGCTTGATCCTGGAGGTCTTTTCTAGTCTGAGCTTCTTTAGCTAGGCTAAAACACCTTGGCTTGTTATTGCCTCTACTTTGATTCTGATAATGCTCACTTGGTCCTACCTATTATCCTTCTACTTGTCCAGTTCAATAAGAAATAAGGACAAGCCTAACTTCATAGTAACCTCTCTATTTTAATCAGTTGTTTAATAATTTACAGGTTCTTAGGCTCCATCCTGTTTGTATGAAATTATAATCTGTGGATTGGCCTTTAAGCCTGCATTCTTAACAAACTCTTCAGTTAATTCTTAGATACACTAAAAATCTGAAGAAACTCTACATGTAACTATTTCTTCAGAGTTTGTCATATACTGCTTGTCATCTGCATGTCTACTCAGCATTTGATTAACATTTGTGTAATAAGAAATAAAATTACACAGTAAGTCATTTAACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA(......ggtctgtaggaaaaacgactattgattgggttagcgtcctaatcgagtatgtggttctgtggctgcaacacagatgtccacagtgacaaggacatgaacacctggatgaacgcgtctgtcaagtctgggtgggctgcatcagtgcctttgcctgtcctgtctcttgcctaagccctcctggttctgactgctcctgcctgggtccctccttcacctgaactctgcaggctgcacagacatgctttctgtatctgtggcccttcattgtccctttccgtg tca......

[0179] TABLE 2 Human −109CCGCCAGTGTGATGGATATCTGCAGAATTCGGCTTACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGTGTGC−3CAGTCACCTTCAGTTTCTGGAGCTGGCCGTCAACATGTCCTTTCCTAAGGCGCCCTTGAAACGATTCAATGACCC42TTCTGGTTGTGCACCATCTCCAGGTGCTTATGATGTTAAAACTTTAGAAGTATTGAAAGGACCAGTATCCTTTCA117GAAATCACAAAGATTTAAACAACAAAAAGAATCTAAACAAAATCTTAATGTTGACAAAGATACTACCTTGCCTGC192TTCAGCTAGAAAAGTTAAGTCTTCGGAATCAAAGAAGGAATCTCAAAAGAATGATAAAGATTTGAAGATATTAGA267GAAAGAGATTCGTGTTCTTCTACAGGAACGTGGTGCCCAGGACAGGCGGATCCAGGATCTGGAAACTGAGTTGGA342AAAGATGGAAGCAAGGCTAAATGCTGCACTAAGGGAAAAAACATCTCTCTCTGCAAATAATGCTACACTGGAAHuman 415AAACAACTTATTGAATTGACCAGGACTAATGAACTACTAAAATCTAAGTTTTCTGAAAATGGTAACCAGAAGAAT|  | |      | |      |   || |     ||     ||   ||||||||||| ||||| |||| ||||||Mouse −75AGGCTAAAGGAGGCAGAATAGATATCTGAGTTCTTATGTTTATTGTAGTTTTCTGAAGATGGTCACCAAAAGAATHuman 490TTGAGAATTCTAAGCTTGGAGTTGATGAAACTTAGAAACAAAAGAGAAACAAAGATGAGGGGTATGATGGCTAAG |||||  ||||||| |||| ||||||||||| ||||| || ||||| |||||||||||| |||||||||  ||MouseATGAGAGCTCTAAGCCTGGAATTGATGAAACTCAGAAATAAGAGAGAGACAAAGATGAGGAGTATGATGGTCAAAHuman 565CAAGAAGGCATGGAGATGAAGCTGCAGGTCACCCAAAGGAGTCTCGAAGAGTCTCAAGGGAAAATAGCCCAACTG|| |||||||||||| |||||||||||| ||| || | |   |||   |||||| | || ||||||| ||| |||Mouse 76CAGGAAGGCATGGAGCTGAAGCTGCAGGCCACTCAGAAGGACCTCACGGAGTCTAAGGGAAAAATAGTCCAGCTGHuman 640GAGGGAAAACTTGTTTCAATAGAGAAAGAAAACATTGATGAAAAATCTGAAACAGAAAAACTCTTGGAATACATC|||||||| |||||||||||||||||||||||||| |||||||||| ||||||||||||||||| ||||||||||Mouse 151GAGGGAAAGGTTGTTTCAATAGAGAAAGAAAAGATCGATGAAAAATGTGAAACAGAAAAACTCTTAGAATACATCHuman 715GAAGAAATTAGTTGTGCTTCAGATCAAGTGGAAAAATACAAGCTAGATATTGCCCAGTTAGAAGAAAATTTGAAA |||||||||| ||||| || |||||||||||||||| |||  ||||||||||||||||||||||| ||||||||Mouse 226CAAGAAATTAGCTGTGCATCTGATCAAGTGGAAAAATGCAAAGTAGATATTGCCCAGTTAGAAGAAGATTTGAAAHuman 790GAGAAGAATGATGAAATTTTAAGCCTTAAGCAGTCTCTTGAGGAAAATATTGTTATATTATCTAAACAAGTAGAA|||||| ||  ||| |||||||| ||||||||||||||||||||||| |||   | ||| ||||| ||| |||||Mouse 301GAGAAGGATCGTGAGATTTTAAGTCTTAAGCAGTCTCTTGAGGAAAACATT---ACATTTTCTAAGCAAATAGAAHuman 875GATCTAAATGTGAAATGTCAGCTGCTTGAAAAAGAAAAAGAAGACCATGTCAACAGGAATAGAGAACACAACGAA|| || | ||| ||||| ||||| ||||||| ||||| |||  ||| ||||| || | ||||||||      |||Mouse 373GACCTGACTGTTAAATGCCAGCTACTTGAAACAGAAAGAGACAACCTTGTCAGCAAGGATAGAGAAAGGGCTGAAHuman 940AATCTAAATGCAGAGATGCAAAACTTAAAACAGAAGTTTATTCTTGAACAACAGGAACATGAAAAGCTTCAACAA| ||| | ||| |||||||||| | | | | ||| | |   ||| |||   |  ||| |||||||||| ||||||Mouse 448ACTCTCAGTGCTGAGATGCAGATCCTGACAGAGAGGCTGGCTCTGGAAAGGCAAGAATATGAAAAGCTGCAACAAHuman 1015AAAGAATTACAAATTGATTCACTTCTGCAACAAGAGAAAGAATTATCTTCGAGTCTTCATCAGAAGCTCTGTTCT|||||||| ||||   | ||||||||||| |||||||| ||| | ||| |  |||| || ||| ||||||| |||523AAAGAATTGCAAAGCCAGTCACTTCTGCAGCAAGAGAAGGAACTGTCTGCTCGTCTGCAGCAGCAGCTCTGCTCTHuman 1090TTTCAAGAGGAAATGGTTAAAGAGAAGAATCTGTTTGAGGAAGAATTAAAGCAAACACTGGATGAGCTTGATAAA|| ||||||||||||      ||||||||  | ||| | |||||  ||||||     |||| |||| | |||Mouse 598TTCCAAGAGGAAATGACTTCTGAGAAGAACGTCTTTAAAGAAGAGCTAAAGCTCGCCCTGGCTGAGTTGGATGCGHuman 1165TTACAGCAAAAGGAGGAACAAGCTGAAAGGCTGGTCAAGCAATTGGAAGAGGAAGCAAAATCTAGAGCTGAAGAA | ||||| |||||||| ||   |||||||||||| || ||  |||||||||||   || || |  || ||| ||Mouse 673GTCCAGCAGAAGGAGGAGCAGAGTGAAAGGCTGGTTAAACAGCTGGAAGAGGAAAGGAAGTCAACTGCAGAACAAHuman 1240TTAAAACTCCTAGAAGAAAAGCTGAAAGGGAAGGAGGCTGAACTGGAGAAAAGTAGTGCTGCTCATACCCAGGCC | |  |  || ||  |   ||||| || ||| || | |||||||||||||  || |||||||||  |||| |||Mouse 748CTGACGCGGCTGGACAACCTGCTGAGAGAGAAAGAAGTTGAACTGGAGAAACATATTGCTGCTCACGCCCAAGCCHuman 1315ACCCTGCT-------------------------------------------------------------------| | || | Mouse 823ATCTTGATTGCACAAGAGAAGTATAATGACACAGCACAGAGTCTGAGGGACGTCACTGCTCAGTTGGAAAGTGTGHuman---------------------------------------------------------------------------Mouse 898CAAGAGAAGTATAATGACACAGCACAGAGTCTGAGGGACGTCACTGCTCAGTTGGAAAGTGAGCAAGAGAAGTACHuman---------------------------------------------------------------------------Mouse 973AATGACACAGCACAGAGTCTGAGGGACGTCACTGCTCAGTTGGAAAGTGAGCAAGAGAAGTACAATGACACAGCAHuman 1323-----------------------------------TTTGCAGGAAAAGTATGACAGTATGGTGCAAAGCCTTGAA                                          | |||| || |||||  |    |  |  || || ||MouseCAGAGTCTGAGGGACGTCACTGCTCAGTTGGAAAGTGTGCAAGAGAAGTACAATGACACAGCACAGAGTCTGAGGHuman 1363GATGTTACTGCTCAATTTGAAAGCTATAAAGCGTTAACAGCCAGTGAGATAGAAGATCTTAAGCTGGAGAACTCA|| ||   |||||| || |||||||||||    | ||||      || |||||||| ||||| ||||||||Mouse 1123GACGTCAGTGCTGAGTTGGAAAGCTATAAGTCATCAACACTTAAAGAAATAGAAGATCTTAAACTGGAGAATTTGHuman 1438TCATTACAGGAAAAAGCGGCCAAGGCTGGGAAAAATGCAGAGGATGTTCAGCATCAGATTTTGGCAACTGAGAGC    |||| |||||||  || | |||||  |||| || ||| |||||||| || ||||| ||| || ||||||||Mouse 1198ACTCTACAAGAAAAAGTAGCTATGGCTGAAAAAAGTGTAGAAGATGTTCAACAGCAGATATTGACAGCTGAGAGCHuman 1513TCAAATCAAGAATATGTAAGGATGCTTCTAGATCTGCAGACCAAGTCAGCACTAAAGGAAACAGAAATTAAAGAA ||||||||||||||| ||||||| ||| |||||| ||  ||| |  |||| |||| |||  |||||||||||||Mouse 1273ACAAATCAAGAATATGCAAGGATGGTTCAAGATTTGCAGAACAGATCAACCTTAAAAGAAGAAGAAATTAAAGAAHuman 1588ATCACAGTTTCTTTTCTTCAAAAAATAACTGATTTGCAGAACCAACTCAAGCAACAGGAGGAAGACTTTAGAAAA||||||  ||| |||||| | ||||||||||||||| | || |||||||  ||||| || ||||||||||| ||ATCACATCTTCATTTCTTGAGAAAATAACTGATTTGAAAAATCAACTCAGACAACAAGATGAAGACTTTAGGAAGHuman 1663CAGCTGGAAGATGAAGAAGGAAGAAAAGCTGAAAAAGAAAATACAACAGCAGAATTAACTGAAGAAATTAACAAG|||||||||||  ||| |  ||||| ||| || |||||||||  ||   ||||||||||    |||||||| ||Mouse 1423CAGCTGGAAGAGAAAGGAAAAAGAACAGCAGAGAAAGAAAATGTAATGACAGAATTAACCATGGAAATTAATAAAHuman 1738TGGCGTCTCCTCTATGAAGAACTATATAATAAAACAAAACCTTTTCAGCTACAACTAGATGCTTTTGAAGTAGAA||||||||||| ||||||||||||||| | ||||| ||||||||||||| |||||| ||||| |||||||  ||TGGCGTCTCCTATATGAAGAACTATATGAAAAAACTAAACCTTTTCAGCAACAACTGGATGCCTTTGAAGCCGAGHuman 1813AAACAGGCATTGTTGAATGAACATGGTGCAGCTCAGGAACAGCTAAATAAAATAAGAGATTCATATGCTAAATTA|||||||||||||||||||||||||||||||||||||| |||||||||||||| ||||| || |||||     ||Mouse 1573AAACAGGCATTGTTGAATGAACATGGTGCAACTCAGGAGCAGCTAAATAAAATCAGAGACTCCTATGCACAGCTAHuman 1888TTGGGTCATCAGAATTTGAAACAAAAAATCAAGCATGTTGTGAAGTTGAAAGATGAAAATAGCCAACTCAAATCG | ||||| |||||  | || ||||||||||| ||||||||||| ||||||||||||||||||||||||||||||Mouse 1648 CTTGGTCACCAGAACCTAAAGCAAAAAATCAAACA TGTTGTGAAATTGAAAGATGAAAATAGCCAACTCAAATCG Human 1963GAAGTATCAAAACTCCGCTGTCAGCTTGCTAAAAAAAAACAAAGTGAGACAAAACTTCAAGAGGAATTGAATAAAGAGGTGTCAAAACTCCGATCTCAGCTTGTTAAAAG GAAACAAAAATGAGCTCAGACTTCAGGGAGAATTAGATAAA Human 2038GTTCTAGGTATCAAACACTTTGATCCTTCAAAGGCTTTTCATCATGAAAGTAAAGAAAATTTTGCCCTGAAGACC| ||| || |||| ||||||||| ||||| |||||||||  ||||| |  ||| || ||||||         ||Mouse 1798GCTCTGGGCATCAGACACTTTGACCCTTCCAAGGCTTTTTGTCATGCATCTAAGGAGAATTTT---------ACTHuman 2113CCATTAAAAGAAGGCAATACAAACTGTTACCGAGCTCCTATGGAGTGTCAAGAATCATGGAAGTAAACATCTGAG|||||||||||||||||  ||||||| | | ||| ||  ||| |  |||||||||||||||||| || |||||Mouse 1873CCATTAAAAGAAGGCAACCCAAACTGCTGCTGAGTTCAGATGCAACTTCAAGAATCATGGAAGTATACGTCTGAAHuman 2188AAACCTGTTGAAGATTATTTCATTCGTCTTGTTGTTATTGATGTTGCTGTTATTATATTTGACATGGGTATTTTA ||  |||||||||||||||  ||| |  |  |  |  |  | |      ||||   |||    ||  || | |Mouse 1948ATACTTGTTGAAGATTATTTTCTTCATTGTTCTTGTTATAGTATATAATGPATTTAATTTCTACTGCGTAGTCTTHuman 2023TAATGTTGTATTTAATTTTAACTGCCAATCCTTAAATATGTGAAAGGAACATTTTTTTTCCAAGTGTCTTTTGAC   | |   |     |  |             | Mouse 2023AGGTATATGAAACGGTAATTCAGCATTTGTTCTCT----------------------------------------Human 2338ATTTTATTTTTTCTTGCAAATACCTCCTCCCTAATGCTCACCTTTATCACCTCATTCTGAACCCTTTCGCTGGCTHuman 2413ATTTTATTTTTTCTTGCAAATACCTCCTCCCTAATGCTCACCTTTATCACCTCATTCTGAACCCTTTCGCTGGCTHuman 2488TCAAGAGTCTAAACCCCAGATTCTTCAGCTTGATCCTGGAGGCTTTTCTAGTCTGAGCTTCTTTAGCTAGGCTAAHuman 2563AACACCTTGGCTTGTTATTGCCTCTACTTTGATTCTTGATAATGCTCACTTGGTCCTACCTATTATCCTTTCTACHuman 2638TTGTCCAGTTCAAATAAGAAATAAGGACAAGCCTAACTTCATAGTAACCTCTCTATTTTAATCAGTTGTTTAATAHuman 2713ATTTACAGGTTCTTAGGCTCCATCCTGTTTGTATGAAATTATAATCTGTGGATTGGCCTTTAAGCCTGCATTCTTHuman 2788AACAAACTCTTCAGTTAATTCTTAGATACACTAAAAATCTGAAGAAACTCTACATGTAACTATTTCTTCAGAGTTHuman 2863TGTCATATACTGCTTGTCATCTGCATGTCTACTCAGCATTTGATTAACATTTGTGTAATAAGAAATAAAATTACAHuman 2938CAGTAAGTCATTTAACCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

[0180] TABLE 3 Human −MSFPKAPLKRENDPSGCAPSPGAYDVKTLEVLKGPVSFQKSQRFKQQKESKQNLNVDKDTTLPASARKVKSSESKHuman 76KESQKNDKDLKILEKEIRVLLQERGAQDRRIQDLETELEKMEARLNAALREKTSLSANNATLEKQLIELTRTNELHuman 151LKSKESENGNQKNLRILSLELMKLRNKRETKMRGMMAKQEGMEMKLQVTQRSLEESQGKIAQLEGKLVSIEKEKI             |+|||||||||||||||||   | ||||||+||| ||+ |  |  || ||||||||||||||Mouse 1             MRALSLELMKLRNKRETKMRSMMVKQEGMELKLQATQKDLTESKGKIVQLEGKLVSIEKEKIHuman 226DEKSETEKLLEYIEEISCASDQVEKYKLDIAQLEENLKEKNDEILSLKQSLEENIVILSKQVEDLNVKCQLLEKE|||+||||||||| |||||||||||+ +||||||| ||||  ||||||||||||| + ||| ||| |||||||Mouse 63DEKCETEKLLEYIQEISCASDQVEKCKVDIAQLEEDLKEKDREILSLKQSLEENITF-SKQIEDLTVKCQLLETEHuman 301KEDHVNRNREHNENLNAEMQNLKQKFILEQQEHEKLQQKELQIDSLLQQEKELSSSLHQKLCSFQEEMVKEKNLF++  |++ ||+ |+ +|||| | ++++||+|| |||||||||  ||||||||||+ | |+||||||||  |||+|Mouse 138RDNLVSKDRERAETLSAEMQILTERLALERQEYEKLQQKELQSQSLLQQEKELSARLQQQLCSFQEEMTSEKNVFHuman 376EEELKQTLDELDKLQQKEEQAERLVKQLEEEAKSRAEELKLLEEKLKGKEAELEKSSAAHTQATLLL-------- ||||  | ||| +||||||+|||||||||| || ||+|  |+  |+ || ||||  ||| || + Mouse213KEELKLALAELDAVQQKEEQSERLVKQLEEERKSTAEQLTRLDNLLREKEVELEKHIAAHAQAILIAQEKYNDTAHuman---------------------------------------------------------------------------Mouse 288QSLRDVTAQLESVQEKYNDTAQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESEQEKYNDTAQSLRDVTAQLESHuman 443QEKYDSMVQSlEDVTAQFESYKALTASEIEDLKLENSSLQEKAAKAGKNAEDVQHQILATESSNQEYVRMLLDLQ||||+    || ||||| ||||+ |  ||||||||| +|||| | | |+ |||| ||   | +|||| ||+ |||Mouse 363QEKYNDTAQSLRDVTAQLESYKSSTLKEIEDLKLENLTLQEKVAMAEKSVEDVQQQILTAESTNQEYARMVQDLQHuman: 518TKSALKETEIKEITVSFLQKITDLQNQLKQQEEDFRKQLEDEEGRKAEKENTTAELTEEINKWRLLYEELYNKTK +| ||| |||||| |||+|||||+|||+||+||||||||++  | |||||   ||| ||||||||||||| |||Mouse: 438NRSTLKEEEIKEITSSFLEKITDLKNQLRQQDEDFRKQLEEKGKRTAEKENVMTELTMEINKWRLLYEELYEKTKHuman 593PFQLQLDAFEVEKQALLNEHGAAQEQLNKIRDSYAKLLGHQNLKGKIKAVVKLKDENSQLKSEVSKLRCQLAKKK||| |||||| ||||||||||| ||||||||||||+||||||||||||||||||||||||||||||||+|| |+|Mouse: 513PFQQQLDAFEAEKQALLNEHGATQEQLNKIRDSYAQLLGHQNLKGKIKHVVKLKDENSQLKSEVSKLRSQLVKRKHuman: 668 QAETKLQEELNKVLGIKHFDPSKAFHHESKENFALKTPLKEGNTNCYRAPMECQESWK*|+|++|| ||+| |||+|||||||| | |||||   ||||||  || Mouse: 588QNELRLQGELDKALGIRHFDPSKAFCHASKENF---TPLKEGNPNCC*

[0181] TABLE 4 Distribution of Staining Scores Among 400 Breast TumorsStaining General Maximum Score Stromal Tumor Nuclear Tumor 0.0 331 21323 3 0.5 35 74 16 24 1.0 18 73 10 28 1.5 8 92 5 54 2.0 3 86 18 69 2.5 434 2 70 3.0 1 17 14 72 3.5 0 3 4 54 4.0 0 0 8 26

[0182] TABLE 5 Univariate analysis of prognostic indicators formetastasis-free and overall survival Metastasis-Free Survival OverllSurvival Num- Num- Factor ber 5-yr Surv p-value ber 5-yr Surv p-valueNodal status None positive 179 83% 186 80% >1 positive 162 50% <0.0001179 60% <0.0001 Tumor size (cm) 2 181 70% 199 72% 2.01-5 149 69% 16069% >5 22 40% 0.03 26 58% 0.01 Tumor grade 1 or 2 292 70% 235 72% 3 7162% 0.74 155 64% 0.53 ER status Negative 23 74% 24 70% Positive 173 74%0.75 187 78% 0.67 Age <50 96 65% 103 68% 50 265 65% 0.75 293 68% 0.19Stromal stain None 301 70% 329 70% Some 62 54% 0.20 69 28% 0.13 Generalstain 0-0.5 95 70% 95 72% 1-1.5 152 58% 165 62% >2 126 68% 0.17 138 70%0.15 Nuclear stain None 292 72% 322 68% Some 71 62% 0.74 76 68% 0.64

[0183] TABLE 6 Multivariate analysis of prognostic factors formetastasis-free and overall survival Metastasis-Free Survival OverallSurvival Factor Odds Ratio p. value Odds Ratio P. value Nodal status2.96¹ <0.0001 2.14¹ <0.0001 Max-Gen RHAMM 1.40² <0.016 1.59² <0.008Tumor Size <0.01 <0.004 2-5 cm 1.25³ <0.02 1.46³ <0.08 >5 cm 1.99³<0.003 1.61³ <0.002

[0184] TABLE 7 RHAMM mRNA expression and tumor grade RHAMM isoform CaseNumber Median Grade p value* RHAMMv4 −/+ 44 6 ++ 54 7 0.0466 RHAMMv4(−9) − 70 7 + 28 8 0.0163 Both isoforms −/+ 40 6 ++/+++ 54 7 0.0357

[0185] TABLE 8 RHAMM mRNA expression and prognostic parameters* RHAMMPoor Prognosis Good Prognosis isoform (12 cases) (15 cases) p value**RHAMMv4 −/+  2 (17%) 11 (73%) ++ 10 (83%)  4 (27%) 0.0063 RHAMMv4 (−9) − 5 (42%) 14 (93%) +  7 (58%)  1 (7%) 0.0085 Both isoforms −/+  3 (25%)11 (73%) ++/+++  9 (75%)  4 (27%) 0.0213

[0186]

1 52 11 amino acids amino acid single linear peptide 1 Lys Gln Lys IleLys His Val Val Lys Leu Lys 1 5 10 10 amino acids amino acid singlelinear peptide 2 Lys Leu Arg Ser Gln Leu Val Lys Arg Lys 1 5 10 3114base pairs nucleic acid single linear cDNA 3 CCGCCAGTGT GATGGATATCTGCAGAATTC GGCTTACTCA CTATAGGGCT CGAGCGGCCG 60 CCCGGGCAGG TGTGCCAGTCACCTTCAGTT TCTGGAGCTG GCCGTCAACA TGTCCTTTCC 120 TAAGGCGCCC TTGAAACGATTCAATGACCC TTCTGGTTGT GCACCATCTC CAGGTGCTTA 180 TGATGTTAAA ACTTTAGAAGTATTGAAAGG ACCAGTATCC TTTCAGAAAT CACAAAGATT 240 TAAACAACAA AAAGAATCTAAACAAAATCT TAATGTTGAC AAAGATACTA CCTTGCCTGC 300 TTCAGCTAGA AAAGTTAAGTCTTCGGAATC AAAGAAGGAA TCTCAAAAGA ATGATAAAGA 360 TTTGAAGATA TTAGAGAAAGAGATTCGTGT TCTTCTACAG GAACGTGGTG CCCAGGACAG 420 GCGGATCCAG GATCTGGAAACTGAGTTGGA AAAGATGGAA GCAAGGCTAA ATGCTGCACT 480 AAGGGAAAAA ACATCTCTCTCTGCAAATAA TGCTACACTG GAAAAACAAC TTATTGAATT 540 GACCAGGACT AATGAACTACTAAAATCTAA GTTTTCTGAA AATGGTAACC AGAAGAATTT 600 GAGAATTCTA AGCTTGGAGTTGATGAAACT TAGAAACAAA AGAGAAACAA AGATGAGGGG 660 TATGATGGCT AAGCAAGAAGGCATGGAGAT GAAGCTGCAG GTCACCCAAA GGAGTCTCGA 720 AGAGTCTCAA GGGAAAATAGCCCAACTGGA GGGAAAACTT GTTTCAATAG AGAAAGAAAA 780 GATTGATGAA AAATCTGAAACAGAAAAACT CTTGGAATAC ATCGAAGAAA TTAGTTGTGC 840 TTCAGATCAA GTGGAAAAATACAAGCTAGA TATTGCCCAG TTAGAAGAAA ATTTGAAAGA 900 GAAGAATGAT GAAATTTTAAGCCTTAAGCA GTCTCTTGAG GAAAATATTG TTATATTATC 960 TAAACAAGTA GAAGATCTAAATGTGAAATG TCAGCTGCTT GAAAAAGAAA AAGAAGACCA 1020 TGTCAACAGG AATAGAGAACACAACGAAAA TCTAAATGCA GAGATGCAAA ACTTAAAACA 1080 GAAGTTTATT CTTGAACAACAGGAACATGA AAAGCTTCAA CAAAAAGAAT TACAAATTGA 1140 TTCACTTCTG CAACAAGAGAAAGAATTATC TTCGAGTCTT CATCAGAAGC TCTGTTCTTT 1200 TCAAGAGGAA ATGGTTAAAGAGAAGAATCT GTTTGAGGAA GAATTAAAGC AAACACTGGA 1260 TGAGCTTGAT AAATTACAGCAAAAGGAGGA ACAAGCTGAA AGGCTGGTCA AGCAATTGGA 1320 AGAGGAAGCA AAATCTAGAGCTGAAGAATT AAAACTCCTA GAAGAAAAGC TGAAAGGGAA 1380 GGAGGCTGAA CTGGAGAAAAGTAGTGCTGC TCATACCCAG GCCACCCTGC TTTTGCAGGA 1440 AAAGTATGAC AGTATGGTGCAAAGCCTTGA AGATGTTACT GCTCAATTTG AAAGCTATAA 1500 AGCGTTAACA GCCAGTGAGATAGAAGATCT TAAGCTGGAG AACTCATCAT TACAGGAAAA 1560 AGCGGCCAAG GCTGGGAAAAATGCAGAGGA TGTTCAGCAT CAGATTTTGG CAACTGAGAG 1620 CTCAAATCAA GAATATGTAAGGATGCTTCT AGATCTGCAG ACCAAGTCAG CACTAAAGGA 1680 AACAGAAATT AAAGAAATCACAGTTTCTTT TCTTCAAAAA ATAACTGATT TGCAGAACCA 1740 ACTCAAGCAA CAGGAGGAAGACTTTAGAAA ACAGCTGGAA GATGAAGAAG GAAGAAAAGC 1800 TGAAAAAGAA AATACAACAGCAGAATTAAC TGAAGAAATT AACAAGTGGC GTCTCCTCTA 1860 TGAAGAACTA TATAATAAAACAAAACCTTT TCAGCTACAA CTAGATGCTT TTGAAGTAGA 1920 AAAACAGGCA TTGTTGAATGAACATGGTGC AGCTCAGGAA CAGCTAAATA AAATAAGAGA 1980 TTCATATGCT AAATTATTGGGTCATCAGAA TTTGAAACAA AAAATCAAGC ATGTTGTGAA 2040 GTTGAAAGAT GAAAATAGCCAACTCAAATC GGAAGTATCA AAACTCCGCT GTCAGCTTGC 2100 TAAAAAAAAA CAAAGTGAGACAAAACTTCA AGAGGAATTG AATAAAGTTC TAGGTATCAA 2160 ACACTTTGAT CCTTCAAAGGCTTTTCATCA TGAAAGTAAA GAAAATTTTG CCCTGAAGAC 2220 CCCATTAAAA GAAGGCAATACAAACTGTTA CCGAGCTCCT ATGGAGTGTC AAGAATCATG 2280 GAAGTAAACA TCTGAGAAACCTGTTGAAGA TTATTTCATT CGTCTTGTTG TTATTGATGT 2340 TGCTGTTATT ATATTTGACATGGGTATTTT ATAATGTTGT ATTTAATTTT AACTGCCAAT 2400 CCTTAAATAT GTGAAAGGAACATTTTTTAC CAAAGTGTCT TTTGACATTT TATTTTTTCT 2460 TGCAAATACC TCCTCCCTAATGCTCACCTT TATCACCTCA TTCTGAACCC TTTCGCTGGC 2520 TTTCCAGCTT AGAATGCATCTCATCAACTT AAAAGTCAGT ATCATATTAT TATCCTCCTG 2580 TTCTGAAACC TTAGTTTCAAGAGTCTAAAC CCCAGATTCT TCAGCTTGAT CCTGGAGGCT 2640 TTTCTAGTCT GAGCTTCTTTAGCTAGGCTA AAACACCTTG GCTTGTTATT GCCTCTACTT 2700 TGATTCTTGA TAATGCTCACTTGGTCCTAC CTATTATCCT TTCTACTTGT CCAGTTCAAA 2760 TAAGAAATAA GGACAAGCCTAACTTCATAG TAACCTCTCT ATTTTAATCA GTTGTTTAAT 2820 AATTTACAGG TTCTTAGGCTCCATCCTGTT TGTATGAAAT TATAATCTGT GGATTGGCCT 2880 TTAAGCCTGC ATTCTTAACAAACTCTTCAG TTAATTCTTA GATACACTAA AAATCTGAAG 2940 AAACTCTACA TGTAACTATTTCTTCAGAGT TTGTCATATA CTGCTTGTCA TCTGCATGTC 3000 TACTCAGCAT TTGATTAACATTTGTGTAAT AAGAAATAAA ATTACACAGT AAGTCATTTA 3060 ACCAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAA 3114 725 amino acids amino acidsingle linear protein 4 Met Ser Phe Pro Lys Ala Pro Leu Lys Arg Phe AsnAsp Pro Ser Gly 1 5 10 15 Cys Ala Pro Ser Pro Gly Ala Tyr Asp Val LysThr Leu Glu Val Leu 20 25 30 Lys Gly Pro Val Ser Phe Gln Lys Ser Gln ArgPhe Lys Gln Gln Lys 35 40 45 Glu Ser Lys Gln Asn Leu Asn Val Asp Lys AspThr Thr Leu Pro Ala 50 55 60 Ser Ala Arg Lys Val Lys Ser Ser Glu Ser LysLys Glu Ser Gln Lys 65 70 75 80 Asn Asp Lys Asp Leu Lys Ile Leu Glu LysGlu Ile Arg Val Leu Leu 85 90 95 Gln Glu Arg Gly Ala Gln Asp Arg Arg IleGln Asp Leu Glu Thr Glu 100 105 110 Leu Glu Lys Met Glu Ala Arg Leu AsnAla Ala Leu Arg Glu Lys Thr 115 120 125 Ser Leu Ser Ala Asn Asn Ala ThrLeu Glu Lys Gln Leu Ile Glu Leu 130 135 140 Thr Arg Thr Asn Glu Leu LeuLys Ser Lys Phe Ser Glu Asn Gly Asn 145 150 155 160 Gln Lys Asn Leu ArgIle Leu Ser Leu Glu Leu Met Lys Leu Arg Asn 165 170 175 Lys Arg Glu ThrLys Met Arg Gly Met Met Ala Lys Gln Glu Gly Met 180 185 190 Glu Met LysLeu Gln Val Thr Gln Arg Ser Leu Glu Glu Ser Gln Gly 195 200 205 Lys IleAla Gln Leu Glu Gly Lys Leu Val Ser Ile Glu Lys Glu Lys 210 215 220 IleAsp Glu Lys Ser Glu Thr Glu Lys Leu Leu Glu Tyr Ile Glu Glu 225 230 235240 Ile Ser Cys Ala Ser Asp Gln Val Glu Lys Tyr Lys Leu Asp Ile Ala 245250 255 Gln Leu Glu Glu Asn Leu Lys Glu Lys Asn Asp Glu Ile Leu Ser Leu260 265 270 Lys Gln Ser Leu Glu Glu Asn Ile Val Ile Leu Ser Lys Gln ValGlu 275 280 285 Asp Leu Asn Val Lys Cys Gln Leu Leu Glu Lys Glu Lys GluAsp His 290 295 300 Val Asn Arg Asn Arg Glu His Asn Glu Asn Leu Asn AlaGlu Met Gln 305 310 315 320 Asn Leu Lys Gln Lys Phe Ile Leu Glu Gln GlnGlu His Glu Lys Leu 325 330 335 Gln Gln Lys Glu Leu Gln Ile Asp Ser LeuLeu Gln Gln Glu Lys Glu 340 345 350 Leu Ser Ser Ser Leu His Gln Lys LeuCys Ser Phe Gln Glu Glu Met 355 360 365 Val Lys Glu Lys Asn Leu Phe GluGlu Glu Leu Lys Gln Thr Leu Asp 370 375 380 Glu Leu Asp Lys Leu Gln GlnLys Glu Glu Gln Ala Glu Arg Leu Val 385 390 395 400 Lys Gln Leu Glu GluGlu Ala Lys Ser Arg Ala Glu Glu Leu Lys Leu 405 410 415 Leu Glu Glu LysLeu Lys Gly Lys Glu Ala Glu Leu Glu Lys Ser Ser 420 425 430 Ala Ala HisThr Gln Ala Thr Leu Leu Leu Gln Glu Lys Tyr Asp Ser 435 440 445 Met ValGln Ser Leu Glu Asp Val Thr Ala Gln Phe Glu Ser Tyr Lys 450 455 460 AlaLeu Thr Ala Ser Glu Ile Glu Asp Leu Lys Leu Glu Asn Ser Ser 465 470 475480 Leu Gln Glu Lys Ala Ala Lys Ala Gly Lys Asn Ala Glu Asp Val Gln 485490 495 His Gln Ile Leu Ala Thr Glu Ser Ser Asn Gln Glu Tyr Val Arg Met500 505 510 Leu Leu Asp Leu Gln Thr Lys Ser Ala Leu Lys Glu Thr Glu IleLys 515 520 525 Glu Ile Thr Val Ser Phe Leu Gln Lys Ile Thr Asp Leu GlnAsn Gln 530 535 540 Leu Lys Gln Gln Glu Glu Asp Phe Arg Lys Gln Leu GluAsp Glu Glu 545 550 555 560 Gly Arg Lys Ala Glu Lys Glu Asn Thr Thr AlaGlu Leu Thr Glu Glu 565 570 575 Ile Asn Lys Trp Arg Leu Leu Tyr Glu GluLeu Tyr Asn Lys Thr Lys 580 585 590 Pro Phe Gln Leu Gln Leu Asp Ala PheGlu Val Glu Lys Gln Ala Leu 595 600 605 Leu Asn Glu His Gly Ala Ala GlnGlu Gln Leu Asn Lys Ile Arg Asp 610 615 620 Ser Tyr Ala Lys Leu Leu GlyHis Gln Asn Leu Lys Gln Lys Ile Lys 625 630 635 640 His Val Val Lys LeuLys Asp Glu Asn Ser Gln Leu Lys Ser Glu Val 645 650 655 Ser Lys Leu ArgCys Gln Leu Ala Lys Lys Lys Gln Ser Glu Thr Lys 660 665 670 Leu Gln GluGlu Leu Asn Lys Val Leu Gly Ile Lys His Phe Asp Pro 675 680 685 Ser LysAla Phe His His Glu Ser Lys Glu Asn Phe Ala Leu Lys Thr 690 695 700 ProLeu Lys Glu Gly Asn Thr Asn Cys Tyr Arg Ala Pro Met Glu Cys 705 710 715720 Gln Glu Ser Trp Lys 725 2123 base pairs nucleic acid single linearcDNA 5 AGGCTAAAGG AGGCAGAATA GATATCTGAG TTCTTATGTT TATTGTAGTT TTCTGAAGAT60 GGTCACCAAA AGAATATGAG AGCTCTAAGC CTGGAATTGA TGAAACTCAG AAATAAGAGA 120GAGACAAAGA TGAGGAGTAT GATGGTCAAA CAGGAAGGCA TGGAGCTGAA GCTGCAGGCC 180ACTCAGAAGG ACCTCACGGA GTCTAAGGGA AAAATAGTCC AGCTGGAGGG AAAGCTTGTT 240TCAATAGAGA AAGAAAAGAT CGATGAAAAA TGTGAAACAG AAAAACTCTT AGAATACATC 300CAAGAAATTA GCTGTGCATC TGATCAAGTG GAAAAATGCA AAGTAGATAT TGCCCAGTTA 360GAAGAAGATT TGAAAGAGAA GGATCGTGAG ATTTTAAGTC TTAAGCAGTC TCTTGAGGAA 420AACATTACAT TTTCTAAGCA AATAGAAGAC CTGACTGTTA AATGCCAGCT ACTTGAAACA 480GAAAGAGACA ACCTTGTCAG CAAGGATAGA GAAAGGGCTG AAACTCTCAG TGCTGAGATG 540CAGATCCTGA CAGAGAGGCT GGCTCTGGAA AGGCAAGAAT ATGAAAAGCT GCAACAAAAA 600GAATTGCAAA GCCAGTCACT TCTGCAGCAA GAGAAGGAAC TGTCTGCTCG TCTGCAGCAG 660CAGCTCTGCT CTTTCCAAGA GGAAATGACT TCTGAGAAGA ACGTCTTTAA AGAAGAGCTA 720AAGCTCGCCC TGGCTGAGTT GGATGCGGTC CAGCAGAAGG AGGAGCAGAG TGAAAGGCTG 780GTTAAACAGC TGGAAGAGGA AAGGAAGTCA ACTGCAGAAC AACTGACGCG GCTGGACAAC 840CTGCTGAGAG AGAAAGAAGT TGAACTGGAG AAACATATTG CTGCTCACGC CCAAGCCATC 900TTGATTGCAC AAGAGAAGTA TAATGACACA GCACAGAGTC TGAGGGACGT CACTGCTCAG 960TTGGAAAGTG TGCAAGAGAA GTATAATGAC ACAGCACAGA GTCTGAGGGA CGTCACTGCT 1020CAGTTGGAAA GTGAGCAAGA GAAGTACAAT GACACAGCAC AGAGTCTGAG GGACGTCACT 1080GCTCAGTTGG AAAGTGAGCA AGAGAAGTAC AATGACACAG CACAGAGTCT GAGGGACGTC 1140ACTGCTCAGT TGGAAAGTGT GCAAGAGAAG TACAATGACA CAGCACAGAG TCTGAGGGAC 1200GTCAGTGCTC AGTTGGAAAG CTATAAGTCA TCAACACTTA AAGAAATAGA AGATCTTAAA 1260CTGGAGAATT TGACTCTACA AGAAAAAGTA GCTATGGCTG AAAAAAGTGT AGAAGATGTT 1320CAACAGCAGA TATTGACAGC TGAGAGCACA AATCAAGAAT ATGCAAGGAT GGTTCAAGAT 1380TTGCAGAACA GATCAACCTT AAAAGAAGAA GAAATTAAAG AAATCACATC TTCATTTCTT 1440GAGAAAATAA CTGATTTGAA AAATCAACTC AGACAACAAG ATGAAGACTT TAGGAAGCAG 1500CTGGAAGAGA AAGGAAAAAG AACAGCAGAG AAAGAAAATG TAATGACAGA ATTAACCATG 1560GAAATTAATA AATGGCGTCT CCTATATGAA GAACTATATG AAAAAACTAA ACCTTTTCAG 1620CAACAACTGG ATGCCTTTGA AGCCGAGAAA CAGGCATTGT TGAATGAACA TGGTGCAACT 1680CAGGAGCAGC TAAATAAAAT CAGAGACTCC TATGCACAGC TACTTGGTCA CCAGAACCTA 1740AAGCAAAAAA TCAAACATGT TGTGAAATTG AAAGATGAAA ATAGCCAACT CAAATCGGAG 1800GTGTCAAAAC TCCGATCTCA GCTTGTTAAA AGGAAACAAA ATGAGCTCAG ACTTCAGGGA 1860GAATTAGATA AAGCTCTGGG CATCAGACAC TTTGACCCTT CCAAGGCTTT TTGTCATGCA 1920TCTAAGGAGA ATTTTACTCC ATTAAAAGAA GGCAACCCAA ACTGCTGCTG AGTTCAGATG 1980CAACTTCAAG AATCATGGAA GTATACGTCT GAAATACTTG TTGAAGATTA TTTTCTTCAT 2040TGTTCTTGTT ATAGTATATA ATGTATTTAA TTTCTACTGC CTAGTCTTAG GTATATGAAA 2100CGGTAATTCA GCATTTGTTC TCT 2123 630 amino acids amino acid single linearprotein 6 Met Arg Ala Leu Ser Leu Glu Leu Met Lys Leu Arg Asn Lys ArgGlu 1 5 10 15 Thr Lys Met Arg Ser Met Met Val Lys Gln Glu Gly Met GluLeu Lys 20 25 30 Leu Gln Ala Thr Gln Lys Asp Leu Thr Glu Ser Lys Gly LysIle Val 35 40 45 Gln Leu Glu Gly Lys Leu Val Ser Ile Glu Lys Glu Lys IleAsp Glu 50 55 60 Lys Cys Glu Thr Glu Lys Leu Leu Glu Tyr Ile Gln Glu IleSer Cys 65 70 75 80 Ala Ser Asp Gln Val Glu Lys Cys Lys Val Asp Ile AlaGln Leu Glu 85 90 95 Glu Asp Leu Lys Glu Lys Asp Arg Glu Ile Leu Ser LeuLys Gln Ser 100 105 110 Leu Glu Glu Asn Ile Thr Phe Ser Lys Gln Ile GluAsp Leu Thr Val 115 120 125 Lys Cys Gln Leu Leu Glu Thr Glu Arg Asp AsnLeu Val Ser Lys Asp 130 135 140 Arg Glu Arg Ala Glu Thr Leu Ser Ala GluMet Gln Ile Leu Thr Glu 145 150 155 160 Arg Leu Ala Leu Glu Arg Gln GluTyr Glu Lys Leu Gln Gln Lys Glu 165 170 175 Leu Gln Ser Gln Ser Leu LeuGln Gln Glu Lys Glu Leu Ser Ala Arg 180 185 190 Leu Gln Gln Gln Leu CysSer Phe Gln Glu Glu Met Thr Ser Glu Lys 195 200 205 Asn Val Phe Lys GluGlu Leu Lys Leu Ala Leu Ala Glu Leu Asp Ala 210 215 220 Val Gln Gln LysGlu Glu Gln Ser Glu Arg Leu Val Lys Gln Leu Glu 225 230 235 240 Glu GluArg Lys Ser Thr Ala Glu Gln Leu Thr Arg Leu Asp Asn Leu 245 250 255 LeuArg Glu Lys Glu Val Glu Leu Glu Lys His Ile Ala Ala His Ala 260 265 270Gln Ala Ile Leu Ile Ala Gln Glu Lys Tyr Asn Asp Thr Ala Gln Ser 275 280285 Leu Arg Asp Val Thr Ala Gln Leu Glu Ser Val Gln Glu Lys Tyr Asn 290295 300 Asp Thr Ala Gln Ser Leu Arg Asp Val Thr Ala Gln Leu Glu Ser Glu305 310 315 320 Gln Glu Lys Tyr Asn Asp Thr Ala Gln Ser Leu Arg Asp ValThr Ala 325 330 335 Gln Leu Glu Ser Glu Gln Glu Lys Tyr Asn Asp Thr AlaGln Ser Leu 340 345 350 Arg Asp Val Thr Ala Gln Leu Glu Ser Gln Glu LysTyr Asn Asp Thr 355 360 365 Ala Gln Ser Leu Arg Asp Val Thr Ala Gln LeuGlu Ser Tyr Lys Ser 370 375 380 Ser Thr Leu Lys Glu Ile Glu Asp Leu LysLeu Glu Asn Leu Thr Leu 385 390 395 400 Gln Glu Lys Val Ala Met Ala GluLys Ser Val Glu Asp Val Gln Gln 405 410 415 Gln Ile Leu Thr Ala Glu SerThr Asn Gln Glu Tyr Ala Arg Met Val 420 425 430 Gln Asp Leu Gln Asn ArgSer Thr Leu Lys Glu Glu Glu Ile Lys Glu 435 440 445 Ile Thr Ser Ser PheLeu Glu Lys Ile Thr Asp Leu Lys Asn Gln Leu 450 455 460 Arg Gln Gln AspGlu Asp Phe Arg Lys Gln Leu Glu Glu Lys Gly Lys 465 470 475 480 Arg ThrAla Glu Lys Glu Asn Val Met Thr Glu Leu Thr Met Glu Ile 485 490 495 AsnLys Trp Arg Leu Leu Tyr Glu Glu Leu Tyr Glu Lys Thr Lys Pro 500 505 510Phe Gln Gln Gln Leu Asp Ala Phe Glu Ala Glu Lys Gln Ala Leu Leu 515 520525 Asn Glu His Gly Ala Thr Gln Glu Gln Leu Asn Lys Ile Arg Asp Ser 530535 540 Tyr Ala Gln Leu Leu Gly His Gln Asn Leu Lys Gln Lys Ile Lys His545 550 555 560 Val Val Lys Leu Lys Asp Glu Asn Ser Gln Leu Lys Ser GluVal Ser 565 570 575 Lys Leu Arg Ser Gln Leu Val Lys Arg Lys Gln Asn GluLeu Arg Leu 580 585 590 Gln Gly Glu Leu Asp Lys Ala Leu Gly Ile Arg HisPhe Asp Pro Ser 595 600 605 Lys Ala Phe Cys His Ala Ser Lys Glu Asn PheThr Pro Leu Lys Glu 610 615 620 Gly Asn Pro Asn Cys Cys 625 630 10 aminoacids amino acid single linear peptide 7 Lys Leu Arg Cys Gln Leu Ala LysLys Lys 1 5 10 10 amino acids amino acid single linear peptide 8 Lys LeuArg Ser Gln Leu Ala Lys Arg Lys 1 5 10 155 base pairs nucleic acidsingle linear DNA 9 CCGCCAGTGT GATGGATATC TGCAGAATTC GGCTTACTCACTATAGGGCT CGAGCGGCCG 60 CCCGGGCAGG TGTGCCAGTC ACCTTCAGTT TCTGGAGCTGGCCGTCAACA TGTCCTTTCC 120 TAAGGCGCCC TTGAAACGAT TCAATGACCC TTCTG 155 99base pairs nucleic acid single linear DNA 10 GTTGTGCACC ATCTCCAGGTGCTTATGATG TTAAAACTTT AGAAGTATTG AAAGGACCAG 60 TATCCTTTCA GAAATCACAAAGATTTAAAC AACAAAAAG 99 78 base pairs nucleic acid single linear DNA 11AATCTAAACA AAATCTTAAT GTTGACAAAG ATACTACCTT GCCTGCTTCA GCTAGAAAAG 60TTAAGTCTTC GGAATCAA 78 122 base pairs nucleic acid single linear DNA 12AGAAGGAATC TCAAAAGAAT GATAAAGATT TGAAGATATT AGAGAAAGAG ATTCGTGTTC 60TTCTACAGGA ACGTGGTGCC CAGGACAGGC GGATCCAGGA TCTGGAAACT GAGTTGGAAA 120 AG122 117 base pairs nucleic acid single linear DNA 13 ATGGAAGCAAGGCTAAATGC TGCACTAAGG GAAAAAACAT CTCTCTCTGC AAATAATGCT 60 ACACTGGAAAAACAACTTAT TGAATTGACC AGGACTAATG AACTACTAAA ATCTAAG 117 87 base pairsnucleic acid single linear DNA 14 TTTTCTGAAA ATGGTAACCA GAAGAATTTGAGAATTCTAA GCTTGGAGTT GATGAAACTT 60 AGAAACAAAA GAGAAACAAA GATGAGG 87 101base pairs nucleic acid single linear DNA 15 GGTATGATGG CTAAGCAAGAAGGCATGGAG ATGAAGCTGC AGGTCACCCA AAGGAGTCTC 60 GAAGAGTCTC AAGGGAAAATAGCCCAACTG GAGGGAAAAC T 101 75 base pairs nucleic acid single linear DNA16 TGTTTCAATA GAGAAAGAAA AGATTGATGA AAAATCTGAA ACAGAAAAAC TCTTGGAATA 60CATCGAAGAA ATTAG 75 179 base pairs nucleic acid single linear DNA 17TTGTGCTTCA GATCAAGTGG AAAAATACAA GCTAGATATT GCCCAGTTAG AAGAAAATTT 60GAAAGAGAAG AATGATGAAA TTTTAAGCCT TAAGCAGTCT CTTGAGGAAA ATATTGTTAT 120ATTATCTAAA CAAGTAGAAG ATCTAAATGT GAAATGTCAG CTGCTTGAAA AAGAAAAAG 179 149base pairs nucleic acid single linear DNA 18 AAGACCATGT CAACAGGAATAGAGAACACA ACGAAAATCT AAATGCAGAG ATGCAAAACT 60 TAAAACAGAA GTTTATTCTTGAACAACAGG AACATGAAAA GCTTCAACAA AAAGAATTAC 120 AAATTGATTC ACTTCTGCAACAAGAGAAA 149 215 base pairs nucleic acid single linear DNA 19GAATTATCTT CGAGTCTTCA TCAGAAGCTC TGTTCTTTTC AAGAGGAAAT GGTTAAAGAG 60AAGAATCTGT TTGAGGAAGA ATTAAAGCAA ACACTGGATG AGCTTGATAA ATTACAGCAA 120AAGGAGGAAC AAGCTGAAAG GCTGGTCAAG CAATTGGAAG AGGAAGCAAA ATCTAGAGCT 180GAAGAATTAA AACTCCTAGA AGAAAAGCTG AAAGG 215 117 base pairs nucleic acidsingle linear DNA 20 GAAGGAGGCT GAACTGGAGA AAAGTAGTGC TGCTCATACCCAGGCCACCC TGCTTTTGCA 60 GGAAAAGTAT GACAGTATGG TGCAAAGCCT TGAAGATGTTACTGCTCAAT TTGAAAG 117 147 base pairs nucleic acid single linear DNA 21CTATAAAGCG TTAACAGCCA GTGAGATAGA AGATCTTAAG CTGGAGAACT CATCATTACA 60GGAAAAAGCG GCCAAGGCTG GGAAAAATGC AGAGGATGTT CAGCATCAGA TTTTGGCAAC 120TGAGAGCTCA AATCAAGAAT ATGTAAG 147 153 base pairs nucleic acid singlelinear DNA 22 GATGCTTCTA GATCTGCAGA CCAAGTCAGC ACTAAAGGAA ACAGAAATTAAAGAAATCAC 60 AGTTTCTTTT CTTCAAAAAA TAACTGATTT GCAGAACCAA CTCAAGCAACAGGAGGAAGA 120 CTTTAGAAAA CAGCTGGAAG ATGAAGAAGG AAG 153 100 base pairsnucleic acid single linear DNA 23 AAAAGCTGAA AAAGAAAATA CAACAGCAGAATTAACTGAA GAAATTAACA AGTGGCGTCT 60 CCTCTATGAA GAACTATATA ATAAAACAAAACCTTTTCAG 100 177 base pairs nucleic acid single linear DNA 24CTACAACTAG ATGCTTTTGA AGTAGAAAAA CAGGCATTGT TGAATGAACA TGGTGCAGCT 60CAGGAACAGC TAAATAAAAT AAGAGATTCA TATGCTAAAT TATTGGGTCA TCAGAATTTG 120AAACAAAAAA TCAAGCATGT TGTGAAGTTG AAAGATGAAA ATAGCCAACT CAAATCG 177 1040base pairs nucleic acid single linear DNA 25 GAAGTATCAA AACTCCGCTGTCAGCTTGCT AAAAAAAAAC AAAGTGAGAC AAAACTTCAA 60 GAGGAATTGA ATAAAGTTCTAGGTATCAAA CACTTTGATC CTTCAAAGGC TTTTCATCAT 120 GAAAGTAAAG AAAATTTTGCCCTGAAGACC CCATTAAAAG AAGGCAATAC AAACTGTTAC 180 CGAGCTCCTA TGGAGTGTCAAGAATCATGG AAGTAAACAT CTGAGAAACC TGTTGAAGAT 240 TATTTCATTC GTCTTGTTGTTATTGATGTT GCTGTTATTA TATTTGACAT GGGTATTTTA 300 TAATGTTGTA TTTAATTTTAACTGCCAATC CTTAAATATG TGAAAGGAAC ATTTTTTACC 360 AAAGTGTCTT TTGACATTTTATTTTTTCTT GCAAATACCT CCTCCCTAAT GCTCACCTTT 420 ATCACCTCAT TCTGAACCCTTTGCTGGCTT TCCAGCTTAG AATGCATCTC ATCAACTTAA 480 AAGTCAGTAT CATATTATTATCCTCCTGTT CTGAAACCTT AGTTTCAAGA GTCTAAACCC 540 CAGATTCTTC AGCTTGATCCTGGAGGTCTT TTCTAGTCTG AGCTTCTTTA GCTAGGCTAA 600 AACACCTTGG CTTGTTATTGCCTCTACTTT GATTCTGATA ATGCTCACTT GGTCCTACCT 660 ATTATCCTTC TACTTGTCCAGTTCAATAAG AAATAAGGAC AAGCCTAACT TCATAGTAAC 720 CTCTCTATTT TAATCAGTTGTTTAATAATT TACAGGTTCT TAGGCTCCAT CCTGTTTGTA 780 TGAAATTATA ATCTGTGGATTGGCCTTTAA GCCTGCATTC TTAACAAACT CTTCAGTTAA 840 TTCTTAGATA CACTAAAAATCTGAAGAAAC TCTACATGTA ACTATTTCTT CAGAGTTTGT 900 CATATACTGC TTGTCATCTGCATGTCTACT CAGCATTTGA TTAACATTTG TGTAATAAGA 960 AATAAAATTA CACAGTAAGTCATTTAACCA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1020 AAAAAAAAAA AAAAAAAAAA1040 264 base pairs nucleic acid single linear DNA (genomic) 26GTGCGTAAGG GGGAAAGAGC TGGGGGGGGG AGACGCCCTA ACGCCCTTTG CCTCTTTCAG 60CTCCCTTCTT GGGAGGCAAG CAGGAGGCGA TTTTAGGGTC GGGCTGGGGC TCATTCAGTT 120GATTGATTTT TCTCAAATAT GCTCTAAGCA TCTGTTACAT GCCAAGCACT AATCAGGATG 180CTAAGGATAC CGCAGTAAAC AGTCTCCGCC CGTGGGCTTA CATTCAGGCG GGGAATACTG 240TCAATAAACA GCGGTAATGG AGAA 264 291 base pairs nucleic acid single linearDNA (genomic) 27 TTCAATCCTT AGTAAGAAAG CCATATATTG CCTGAATATA TGATGTCATCTCAAAACTGC 60 GTTTGCTCAG TTGCCTGTGT TCCTTTGACC CGGTTGATAT AAAGGGCAAGATGATATTGT 120 TCTTCATAGA GAGGCCTTCT TTGTAATATC AAATGGATGC AATTTTTTACATTTAAAAAA 180 AGCAGTTTGT TAATGACATT TTTACATTTA TATTCACTTT ATTATGACATGTTTTAACTT 240 AAGATCATAA GTAACATTAG ATAATATATT AATGTTTTCT ATTTCCTCTA G291 227 base pairs nucleic acid single linear DNA (genomic) 28GTAATAAGAT CACCAAAGAA CAATGGTTAT GTGATCTTAT AAGTTTTAAA GTTATGAATA 60ACAATATTTA AAGATGTTAT AGCATTTTTT AAAATGTGAA GCTAGAACTA TATTTAAATT 120TTATTTGATG GATTTATGAA AGGGTCAAGT ACAGAATAAT GCTGTCATCA TTACATTGTT 180ATATAACCAG GAAAATTAAG CAAGATACTT ATATTGATAT GTAGCTT 227 244 base pairsnucleic acid single linear DNA (genomic) 29 GGTGAGGAGC TTTTATATGCCAGCTGGTTT ATCAAGTGTA TCATCAAAAA CATCTGAAAG 60 TATTGTATTT GATTAGAATGGGTTAAAGTG TATGAATCAA GGTTATAACT AAATCTGTAA 120 ATTAATGAAA TGAGTTATCATTAGAACTCT AGCAAGTTTT ACATTTCTGC CTAGGTCATT 180 ATGTTTAAAT GTGCCCTTAGTTCACAATTA TAATGGTCTT CAATTCTCAA TCACTTCTAT 240 GTTT 244 274 base pairsnucleic acid single linear DNA (genomic) 30 TTATGTTCTT AAAATGCATTAAGACTTTAA GATGTATCAT AGGTAAATAT GATTATTCAA 60 ATAGCTAGTA ACATTAGAATATCTACAAGC ATAATGTCAA AATCAGAGAT TTTTCCAGAA 120 ACTTTAGGGG TGATTATTGGTAGCATCTCC TTATGTTGGC ATTCTATCAG TGAATCATTT 180 ATTATCACCT TGTTTTTGTCCAGATTCGTG TTCTTCTACA GGAACGTGGT GCCCAGGACA 240 GGCGGATCCA GGATCTGGAAACTGAGTTGG AAAG 274 286 base pairs nucleic acid single linear DNA(genomic) 31 GTATCTGAGC CTCATGATAA CATTTACAAT TGAATAAATA TAAACACGTTTTTTAGGGCC 60 GGGCACGGTG GCTCACGCCT GTGTTCCCAG CATTTTGGGA GGCCAAGGCAGGCGGATCAC 120 CTGAGGTCGG GAGTTCGAGA CCAGCCTGAC CAACATGGAG AAACCCTGTCTCTACTAAAA 180 ATACAAAAAT TAGCTAGGCC TATTGGCGGG CGCCTGTAAT CTCAGCTACTCGGGAGGCTG 240 AGGCAGAAGA ATCACTTGAA CCCAGGAGGT GGAGGTTGCA GTGAGC 286521 base pairs nucleic acid single linear DNA (genomic) 32 TAAACCAGCAAGTCACATTA AGGAAAAGAG GGATAAGAAC AGTAGACTGG TACAGTGGCT 60 CATGCCTGTATTTCCAGCAT TTTGGAAGGC TGAGGGCTGG AGAATTGCTT GAGGCCAGGA 120 GTTTGAGACCAGCCTGGGCA ACATATCAAG ACCCCATCTC TATAAACAAA TTGAAAAATT 180 AGCTAGGCATGGTGGTGGTG CACACCGGTA ATCCCAGCTA CTCAGGAAGA TGAGGCAGGA 240 GGATTGATTGAGCCCAGGAG TTTGAGATTA TAGCGAGCTA TGATCATGCC ACTCCACTCT 300 AGCCGTGACAGCGGAGCGAG ACTTGATCTC TTAAAAAGAA AAGAAAAAAA AATTAAATCA 360 ATCAGTAATTATGGTGTAGG TCAAAGACTG TTCTCTCTAC CAAAGTATAT TAAAGTCAAA 420 AACATAACCCCAGTGATAGG TAGAAAAATC AATATTTCTC TATTTTAAAT ATGTCTTAGC 480 AGAAAATATTTCTGAATTTT TTACGTGTTT GTTGTATTTA G 521 91 base pairs nucleic acid singlelinear DNA (genomic) 33 GTGAGTGCTG CCCTTGGCAG GTTTGCTGTG TCTGGATCTGGGGATCAGTA CAACTTTCTC 60 ATTTCCTAAA ACAGGTATCT TTGTTGTGTA G 91 467 basepairs nucleic acid single linear DNA (genomic) 34 GTAAGTGAGT GAATGTGAAGAGAAATTGTT AAGTGGAAGC AATTCTTGAT TTGAGTCTCT 60 TCACAATTAT TGTTTACTAGACTTAACCTT CTCTTAGTAC TTATCTCATT GCCTCCCTCC 120 AGTTGCCCTA TTTCTCTTTTTAAACTAGAA TGAGCCCTAA TCATTCTCAA ACATGTTGTG 180 CTACAAAGTT GTATGAGTGCATTACTTTTG TACATCTTCT GTATTATTAA TGATGAGGAA 240 AGATTTCATG ATCTTATGAAAGTGGTCATT AGATTGAAAT TGAGAAACAC TGGTATAGGA 300 AATTGTGATT TATGCACAATCCTAGCCTTT GATTTTGAGC TTTAATATAC ATATAATAAA 360 ATGTGTGGAT AGTAAGTATTCAGTTTGGTG ACTTTAGCAA TTGTATACAC CTACTAACCA 420 CTACCAAACA AGATAGAACATTTTCATCCC TTCAGAAAGT TCCTTCA 467 549 base pairs nucleic acid singlelinear DNA (genomic) 35 TTCTACTAGG TAGGAAGTGG TATCTCCTTT GTGATTTTAATTTGTTACCA TGAATGTTGA 60 CCTTATTTTT ATGTGCTTAT TGACCATTTT ATGTGCATACAACTTTTGCA AGGTGTCTAT 120 TGAAGTCTTT TGTCCATTTC TTGCATTGGA CAGTTTGGTGGAGGTAAACA GATAAGTAAT 180 TGAAGACCAG GTAGTCTGGG ACAAAAGCTT TATGGGCACACAAAATGCTA TTTAGTATGT 240 TGGATGGGTG GGGAAACCAG GAAGACCACA AAAAGAATATTATTTCTAAC ACTTGGGATA 300 CTGTAATGAA GGTTCTGTCA TCATAGGTTT TTTTGCAGTATATATTCAGA AAACTTTCTC 360 ACTTAAATAA AAATTTTAGT CTTCTATTTT GATGTAAATTGTGATTTGAG AAATTACATA 420 AAATAATAGT TAAGAGTTAG GGCTCTGTAG TCAGCCTGCCTGATACAGGA GTATCTGGTA 480 CATAAGCATT ATGTAAGATT ATTAAATAAC GAAACTAGAATGTATTAACA TATGCAATTT 540 TTGTTTTAG 549 125 base pairs nucleic acidsingle linear DNA (genomic) 36 GTAATATGAG CAGTAGCTTT AAATTGAACCTTATTTTTTT AATACTCAGT CATTTTCATC 60 ATTTTTCTGT TATTTTCCCT GTGCCTAAATAGATGTGCTT TTTAAGATAA TTTGTTTTAA 120 TGCAG 125 497 base pairs nucleicacid single linear DNA (genomic) 37 GTATTACAGT GTTTATAGTT ACTTTGTTTAGATAAGTGTT ACATACAACA TTTAGGAAAA 60 ATACTACTAT GCTAAAACAA CCTTTTAAATATAATTAGCT ATACTAACAT TTTAAATATA 120 ATTAGCTATA TAGCTATACA ACAGCAAAAACCTGTACTGC ATTTTAGAAT ATTTTACTCT 180 TATAATGTTT GTTTTCTGTT TATTTCAATACAGCATATTA CCTGTCTTGA TTGAAATATA 240 TACAGTCATA TAATTCTTGA CTTTCCACTAGGTAGCTGTG TAACAATCAG TAGATAACAC 300 AGAACAAGAT TTGTGGGTTT TATTATTTAGCACATAGTAT ATATTACATG GAGTAATGAT 360 ACAAAGTTCA CAGTTTTGTT TTCTTCTTTGGAAATACCAT GCTAAAAGCA GTGTAATGGA 420 ATATTATGGG AGTCCAGGTT TCTCAGTCTTAATGTTCTTA TCTAATTCCA GTATTCTTGA 480 TGTTTTGAGT TTTCTAG 497 295 basepairs nucleic acid single linear DNA (genomic) 38 GTAATTTACC ACCATATTTTTTTAAACTGT TCATTTTGTG TCATACATTT CCCTATGTCT 60 CTGAACACCT TTAAATTGTGTATATCCTTT GATCTACCAA TTCTATCTTT AGAGTCTTAT 120 CCTGAGGACA TAATCATGGATATGCTGAGG ATTTAGCTAC GTATTTTCAC TACATGTTCA 180 CCTAGGGTTA TGAATAATGTGGGAAATGAC AACAGATACA AAATAGGGAA TTTTTAAAAA 240 ATTTTCTGGC TCATTCTTGTGTTATTTAGG CTATATAAAC ATTACACTTA CCTTG 295 328 base pairs nucleic acidsingle linear DNA (genomic) 39 TAATTTTATG TAATATGGTG TGAAAAATAATGTTAATATC AAAGCCAGTT GTAAAACAGA 60 TATATATATA TAAAAATATA ATTTTAGATTAAGAAGTTTC TGCATGTGCG TTGCATAGAA 120 AAAAGCCTAA GATGATATTT GCCACAATGTTAACAAGGTA TAGGAAATAA TCTATGAAAA 180 CAAATATGCT ATTTCTATAT TGTTTTAAGTTTCCTTGAAT CTGTGGAATT TAGGTTTCAT 240 CCTTCTTTAT CTGTACTTTT TTTTGTCTCCTAGTACAACC TCACAATGCC ATTCCAAATT 300 ATTTTGGTGG TTTTCTGTTT GGATATAG 328112 base pairs nucleic acid single linear DNA (genomic) 40 GTTTGTATTAATAGGATCTC ATGTTTTATT ATGACTTCAG ATGTATTTAT TTTGAGTACT 60 TTTTTTAGTATTCTCTTATC AATCATGTGA GCGTGTTAGG TTGGATTATT TT 112 255 base pairsnucleic acid single linear DNA (genomic) 41 TTATACCTAC TACCTTCTTCACCCAAATTT TTAAAGTAAA ATAAGCAGGA AAGATAAGTT 60 GAAGCTAGTA GAAAAATGCATTAAAAAACA TGCTTTCGAG GTAAGTCATA AATTAGGATC 120 TGAGCTATTT AGCAGGTAATGCAGTGGTGA AGATATGAGC TATATGATTC ACAGTTTCAA 180 AGGTAAATAC TATTTTCTTTCTTAGGGTAG TAATTGTAGG TGGCATTTTA TCTTTCAATT 240 ATTTCTTTTT CTTAG 255 206base pairs nucleic acid single linear DNA (genomic) 42 GTATTTTTCTTGGGAGCCTG CACTCTTAAA TATGATGTGT GCAGAAAGGG GTGTTTACCC 60 CAGGAAATATGTGAGCAAAG CAGTCACACA AAGGATGATT CATACTAGTT TAAATTCCAT 120 AATCACCAACCGTAAGTGGG CATTTAGCAT TATCTGGTAA TCTTATTGTA TTTATATAAT 180 TCCCTTTATAATTTATAGAA ATTCCC 206 207 base pairs nucleic acid single linear DNA(genomic) 43 TTTTTTTTTC TTTGAATACA CAGCAGATGC CATGTAAACT CATTAGTACTTGCCTCAGAA 60 CACTGAATTC TTACCTGTGT TAAATGCATG AATACATTAA AAACTTTTTAGTTTTACTTA 120 GAAGTATATA AAGTGTCCCC TAATCAGTTA TGATTGTCAT ACGCAATAGTTAGAAAACTA 180 CTTTGACTTT TTTTTCTTTT TAATAAG 207 231 base pairs nucleicacid single linear DNA (genomic) 44 GTATATAGAG CAAATAATGG CCTTAGAACCATTAAGACAA TTTAATGTTG AAAGCCAGCT 60 AGTAACTGTC CCTTGGCTTG CTTTTGGCCATCTTATACTG CAAATTAAGA ATTTACTCAG 120 TTAAAAAATG ACACTTCTTG AAGAGTTCCTTGAGGTTTAA AGAAAAAAAA AGGAAAAATT 180 AATGAAAGTG GCTATAAAAT GTTTAGTGACCTCTTCTCTC TCAAACCAAA G 231 95 base pairs nucleic acid single linear DNA(genomic) 45 GTAATCTATG ATTCGAACCT GAGTGCCTTG TTAACTCAGT TACGATGTGATTTTTTAAAT 60 AACTATGTTT TTCTCAATTT AATTCTTCCA TGCAG 95 249 base pairsnucleic acid single linear DNA (genomic) 46 GTTTGTCAGT TAGGAGTAAACTTACTTGTG TTTATTTTAG GGACTCTTTG TTCCCTATTA 60 TAGTGAGGAC AGTGACTCGGGTTTTCTGCA AGATCATTTT GCTCTGCACT TACAGTGCCA 120 ATTTAGCTCA CTATTAAAGGTTTATACATT TTATTAAATT ATGCATAATT TTTTCCCACA 180 TTATTGAAGT ATAATTGACAAATTTAATTG ACATAATTTT TCAATGGACC TTTGTGGTTT 240 TAAAAAAAA 249 151 basepairs nucleic acid single linear DNA (genomic) 47 CTCATAGAGA ATCTATGGAGAGCCCTGAGA ATATGTGAAC ATACCTTGTT TTCATTTGTG 60 TTTTTAATTT TCTTTAGTGTTTATGGTTTA TATGAAACTA GTAAGATCAA ACTGTTTTAA 120 GTCTTAACTT TATTTAAAAAATCTTTTTCA G 151 275 base pairs nucleic acid single linear DNA (genomic)48 GTTTGTAAAA TGACTTTTCA TTTTATTAAA GATATTGGAG TGGGGGTTAT TCTAACTATA 60ATACTTAAAT AAAATGAATA TCTTTGGTAT CAGAAAAAAA TAACTGTTTA TAGAGGAAAA 120TTGAGCTGTG ATTTAGTGGA TTTATTTTAG AGTGTTGACC AGATGGGCAT TCAATGTTCT 180AAAGTTTTCT AGCTACCGTC TTAATATATA TTGAAAATTA CTTGAGTAAA TTTGATGAAT 240TCATTAAGCT TTACATATCT ATTTCCATTT GCAAA 275 282 base pairs nucleic acidsingle linear DNA (genomic) 49 GGTCTGTAGG AAAAACGACT ATTGATTGGGTTAGCGTCCT AATCGAGTAT GTGGTTCTGT 60 GGCTGCAACA CAGATGTCCA CAGTGACAAGGACATGAACA CCTGGATGAA CGCGTCTGTC 120 AAGTCTGGGT GGGCTGCATC AGTGCCTTTGCCTGTCCTGT CTCTTGCCTA AGCCCTCCTG 180 GTTCTGACTG CTCCTGCCTG GGTCCCTCCTTCACCTGAAC TCTGCAGGCT GCACAGACAT 240 GCTTTCTGTA TCTGTGGCCC TTCATTGTCCCTTTCCGTGT CA 282 25 amino acids amino acid single linear peptide 50 ValSer Ile Glu Lys Glu Lys Ile Asp Glu Lys Ser Glu Thr Glu Lys 1 5 10 15Leu Leu Glu Tyr Ile Glu Glu Ile Ser 20 25 25 base pairs nucleic acidsingle linear other nucleic acid /desc = “primer” 51 GGATATCTGCAGAATTCGGC TTACT 25 24 base pairs nucleic acid single linear othernucleic acid /desc = “primer” 52 ACAGCAACAT CAATAACAAC AAGA 24

We claim:
 1. An isolated nucleic acid comprising a nucleotide sequenceencoding a protein selected from the group consisting of human RHAMM 1,human RHAMM 2, human RHAMM 3, human RHAMM 4 and human RHAMM
 5. 2. Theisolated nucleic acid of claim 1 wherein the nucleic acid encodes theamino acid sequence of Sequence ID NO:4.
 3. The isolated nucleic acid ofclaim 2 wherein the nucleotide sequence is selected from the groupconsisting of (a) the genomic sequence of human RHAMM; and (b) thenucleotide sequence of Sequence ID NO:3.
 4. The isolated nucleic acid ofclaim 1 selected from the group consisting of (a) a nucleotide sequencecomprising in continuous sequence the nucleotide sequences of SequenceID NOS:9 to 25; (b) a nucleotide sequence comprising in continuoussequence the nucleotide sequences of Sequence ID NOS:9, 10, 11 and 13 to25; and (c) a nucleotide sequence comprising in continuous sequence thenucleotide sequences of Sequence ID NOS:9, 10 and 12 to
 25. 5. Anisolated nucleic acid comprising a nucleotide sequence selected from thegroup consisting of (a) a nucleotide sequence of at least 10 consecutivenucleotides of Sequence ID NO:3; (b) a nucleotide sequence of at least15 consecutive nucleotides of Sequence ID NO:3; and (c) a nucleotidesequence of at least 20 consecutive nucleotides of Sequence ID NO:3. 6.An isolated nucleic acid comprising a nucleotide encoding at least onebinding domain of human RHAMM protein or a fragment or analogue thereofwhich retains HA binding ability.
 7. The isolated nucleic acid of claim6 encoding the amino acid sequence of Sequence ID NO:1 or Sequence IDNO:7.
 8. An isolated nucleic acid comprising a nucleotide sequenceselected from the group consisting of Sequence ID NO:9, Sequence IDNO:10, Sequence ID NO:11, Sequence ID NO:12, Sequence ID NO:13, SequenceID NO:14, Sequence ID NO:15, Sequence ID NO:16, Sequence ID NO:17,Sequence ID NO:18, Sequence ID NO:19, Sequence ID NO:20, Sequence IDNO:21, Sequence ID NO:22, Sequence ID NO:23, Sequence ID NO:24 andSequence ID NO:25.
 9. The nucleic acid of claim 8 wherein the nucleotidesequence is Sequence ID NO:16.
 10. An isolated nucleic acid comprising anucleotide sequence encoding the amino acid sequence of Sequence IDNO:50.
 11. An isolated nucleic acid comprising a nucleotide sequenceselected from the group consisting of the Sequences ID NO:26 to
 49. 12.A recombinant expression vector comprising an isolated nucleic acid ofany of claims 1 to
 11. 13. A host cell transformed with a recombinantexpression vector of claim
 12. 14. A transgenic animal wherein a genomeof the animal, or of an ancestor thereof, has been modified by insertionof at least one recombinant construct to produce a modification selectedfrom the group consisting of (a) insertion of a nucleotide sequence ofat least one exon of the human RHAMM gene; (b) insertion of a nucleotidesequence encoding at least one human RHAMM protein; (c) inactivation ofan endogenous RHAMM gene.
 15. A substantially pure protein selected fromthe group consisting of human RHAMM 1, human RHAMM 2, human RHAMM 3,human RHAMM 4 and human RHAMM
 5. 16. The protein of claim 15 comprisingthe amino acid sequence of Sequence ID NO:4 or a fragment or analoguethereof which retains the ability to bind hyaluronan.
 17. Asubstantially pure peptide comprising an amino acid sequence selectedfrom the group consisting of (a) at least 5 consecutive amino acidresidues from the amino acid sequence of Sequence ID NO:4; (b) at least10 consecutive amino acid residues from the amino acid sequence ofSequence ID NO:4; and (c) at least 15 consecutive amino acid residuesfrom the amino acid sequence of Sequence ID NO:4.
 18. A substantiallypure peptide comprising at least one binding domain of human RHAMM. 19.A peptide of claim 18 selected from the group consisting of Sequence IDNO:1 and Sequence ID NO:17.
 20. A substantially pure peptide having theamino acid sequence of Sequence ID NO:50.
 21. An antibody whichselectively binds to an antigenic determinant of a human RHAMM protein.22. An antibody which selectively binds to an antigenic determinant ofthe peptide of claim
 20. 23. A cell line producing an antibody of claim21 or
 22. 24. A method for identifying compounds which can selectivelybind to a human RHAMM protein comprising the steps of providing apreparation of at least one human RHAMM protein; contacting thepreparation with a candidate compound; and detecting binding of theRHAMM protein to the candidate compound.
 25. The method of claim 24wherein the binding of the RHAMM protein to the compound is detected bya method selected from the group consisting of affinity chromatography,a yeast two-hybrid system, and a phage display library.
 26. A method forassessing prognosis in a mammal having a tumour, comprising obtaining atumour sample from the mammal and determining the level of expression ofRHAMM protein in the tumour sample, wherein increased expression ofRHAMM protein is indicative of a poor prognosis.
 27. The method of claim26 wherein RHAMM expression is determined by a method selected from thegroup consisting of a histochemical method, a method comprisingdetermination of the level of RHAMM mRNA in a biopsy sample and a methodcomprising determination of expression of human RHAMM exon 8 in a biopsysample.
 28. The method of any of claims 26 to 27 wherein the mammal is ahuman and the tumour is a breast tumour.
 29. A pharmaceuticalcomposition for preventing or treating a disorder in a humancharacterised by overexpression of the RHAMM gene comprising aneffective amount of a nucleotide sequence selected from the groupconsisting of (a) a dominant suppressor mutant of the RHAMM gene; (b) anantisense sequence to human RHAMM cDNA; and (c) an antisense sequence toexon 8 of the human RHAMM gene and a pharmaceutically acceptablecarrier.
 30. A method for preventing or treating a disorder in a humancharacterised by overexpression of the RHAMM gene comprisingadministering to the mammal an effective amount of a nucleotide sequenceselected from the group consisting of (a) a dominant suppressor mutantof the RHAMM gene; (b) an antisense sequence to human RHAMM cDNA; and(c) an antisense sequence to exon 8 of the human RHAMM gene.
 31. themethod of claim 32 wherein the disorder is cancer.
 32. A method forinhibiting cell migration in a human comprising administering to thehuman an effective amount of an agent selected from the group consistingof (a) an antibody which binds specifically to human RHAMM protein or afragment thereof; and (b) a peptide comprising a human RHAMM HA-bindingdomain.